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SureSelect Automated RNA Target Enrichment

1. SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 71 3 Sample Preparation 16 When verification is complete click Run Selected Protocol Run Selected Protocol 17 Running the Pre CapturePCR_RNASeq_ILM_v1 0 pro protocol takes approximately 15 minutes Once complete the PCR ready samples containing prepped DNA and PCR master mix are located in the PCR plate at position 6 of the Bravo deck and you will see the following prompt Plate ready to seal Seal PCR plate and run thermocycler protocol User data entry Pause and Diagnose Continue a Remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds b Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles c Transfer the plate to the thermal cycler with the heated lid ON and run the program in Table 35 72 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Table 35 Thermal cycler program for mRNA Library PCR indexing Segment Number of Cycles Temperature Time 1 1 37 C 15 minutes 2 1 95 C 2 minutes 3 9 13 cycles 95 C 30 seconds i 65 C 30 seconds 72 C 1 minute 4 1 72 C 5 minutes 5 1 4 C Hold Table 36 mRNA Library PCR indexing cycle number recommendations Amoun
2. Run VWorks protocol AMPureXP_v1 1 pro Pre Capture PCR 12 On the SureSelect setup form under Select Protocol to Run select AMPureXP_v1 1 pro Pre Capture PCR 13 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 9 14 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 15 Click Display Initial Workstation Setup Display Initial Workstation Setup 16 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini Anant ee A Shelf 5 rA ShA MOP nO a a saat to a _gae ee a T SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 17 When verification is complete click Run Selected Protocol Run Selected Protocol The purification protocol takes approximately 45 minutes When complete the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7 Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 71 3 78 Sample Preparation Step 6 Assess library DNA quantity and quality Stopping Point Option 1 Analysis using the Agilent 2100 Bioanalyzer
3. SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 23 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation VWorks Automation Control Software 4 The Workstation Setup region of the form will then display the required placement of reaction components and labware in the NGS Workstation for the specified run parameters QA VWorks SureSelect RNA MLV a a a a E Workstation Setup m MiniHub SureSelect RNA MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 for Illumina sequencers Shelf5 Empty Nunc Eset Nunc Empty Nunc DeepWell Plate DeepWell Plate DeepWell Plate Shelf 4 Bead Binding Buffer in twin tec Parameters Shelf 3 Bead Elution 1 Select Protocol to Run Buffer in twin tec mRNA_Purification_v1 0 pro 7 Shelf 2 Empty Tip Box Bead Wash Buffer in Nunc DeepWell AMPureXP Case Not Applicable Shelf 1 New Tip Box Empty Tip Box 2 Select PCR Plate labware for Thermal Cycling 96 ABI PCR half skirt in Red Alum Insert b4 Bravo Deck 3 Select Number of Columns of Samples Fa 4 Click button below to Display Initial Workstation Setup fame Ser lt Position 1 gt _ lt Position 2 gt __ lt Position 3 gt Display Initial Clear Workstation Waste Reservoir Workstation Setup Setup Display Axygen 96DW 5 Load labware according to Workstation Setup gt 4 lt Pos 4 Peltier gt RT
4. 5 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 6 Empty tip box 8 New tip box 9 Prepped library DNA in Eppendorf plate oriented with well A1 in the upper left 6 On the SureSelect setup form under Select Protocol to Run select Aliquot_Libraries_v1 0 pro 7 Click Display Initial Workstation Setup Display Initial Workstation Setup SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 83 4 Hybridization 8 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini Tom Ae Shelf 5 oo ISRA U aai a at ae a el 9 When verification is complete click Run Selected Protocol Run Selected Protocol 10 When prompted by the dialog below browse to the csv file created for the source plate of the current run and then click OK to start the run E Select Hit Pick Input File Please select the hit pick input file for the hit pick replication task at task 5 of subprocess Aliquot Libraries C vWorks Workspace NGS Option B XT_Illumina_1 5 Aliquot Library Input E O ae The library aliquoting protocol takes approximately 1 hour for 96 samples When complete the 100 ng samples are in the PCR plate located on Bravo deck position 5 11 Remove the 100
5. Agilent p n Gb498B 13 Agilent p n 19477 022 SureCycler 8800 Thermal Cycler Agilent p n G8810A 96 well plate module Agilent p n G8810A and compression mats Agilent p n 410187 or equivalent Agilent p n 401334 Eppendorf p n 951020401 or 951020619 Thermo Scientific p n 1064156 Thermo Scientific p n 260251 Axygen p n P 2ML SQ C E amp K Scientific p n EK 2440 Millipore p n 3097 Savant SpeedVac model DNA120 with 96 well plate rotor model RD2MP or equivalent DynaMag 50 magnet Life Technologies p n 123 02D or equivalent SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Table3 Required Equipment continued Before You Begin 1 Description Vendor and part number DNA Analysis Platform and Consumables Agilent 2100 Bioanalyzer Laptop Bundle Agilent 2100 Bioanalyzer Electrophoresis Set Agilent DNA 1000 Kit Agilent High Sensitivity DNA Kit OR Agilent 2200 TapeStation Agilent D1000 ScreenTape Agilent D1000 Reagents Agilent High Sensitivity D1000 ScreenTape Agilent D1000 Reagents DNA LoBind Tubes 1 5 mL PCR clean 250 pieces P10 P20 P200 and P1000 pipettes Ice bucket Powder free gloves Vortex mixer Timer Agilent p n G2943CA Agilent p n G2947CA Agilent p n 5067 1504 Agilent p n 5067 4626 Agilent p n G2964AA or G2965AA Agilent p n 5067 5582 Agilent p n 5067 5583 Agilent p n 5067 5584 Agilent p n 5067 5585 Eppendorf p n 022431021 or equivalent
6. PCR Master Mix Position on Source Plate Column 4 A4 H4 Volume of Master Mix added per Well of Nunc Deep Well Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs 36 7 uL 63 4 uL 90 0 uL 116 7 pL 170 0 pL 343 2 uL 110 If you are using a new DeepWell plate for the post capture PCR source plate for example when amplifying the second half of the captured DNA sample leave columns 1 to 3 empty and add the PCR Master Mix to column 4 of the new plate SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 10 e3 A is co s Se O X eS lt gt 00 ER gt 00 ie oS e e LS FS os amp S LSS S Jy E g ew sS S ee A D N 6 R 7V N e amp S a g 8 Figure 12 Configuration of the master mix source plate for Post CapturePCR_RNASe q_ILM_v1 0 pro Columns 1 3 were used to dispense master mixes for the SureSelectHybridization_v1 0 pro protocol 8 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 9 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 111 5 Indexing and Sample Prep
7. complete two actions during the runset at the time points in the table below The times provided are approximate each action is completed in response to a VWorks prompt at the appropriate time in the runset Table 51 Operator action Approximate time after run start Transfer hybridization reactions from lt 5 minutes thermal cycler to NGS workstation Remove PCR plate from red aluminum 5 10 minutes insert Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a NucleoClean decontamination wipe 3 Pre set the temperature of Bravo deck position 4 to 66 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks Bravo deck position 4 corresponds to CPAC 2 position 1 on the Multi TEC control touchscreen 98 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 Prepare the Dynabeads M 270 streptavidin beads CAUTION Use only the recommended Dynabeads M 270 Streptavidin Beads for this automated protocol Use of other streptavidin bead preparations may adversely affect performance and is not supported by Agilent 4 Vigorously resuspend the Dynabeads M 270 Streptavidin magnetic beads on a vortex mixer The beads settle during storage 5 Wash the magnetic beads a Inaconical vial combine the components li
8. lt Pos 5 Shaker gt lt Pos 6 Peltier gt RT Controls Oligo dT beads Empty Plate Set Set labware in 2 labware in 2 Once you have loaded labware according to Workstation Setup on right click Run Selected Protocol to start run Run Selected lt Pos 7 Magnetic gt lt Position 8 gt lt Pos 9 Chiller gt 0 C Protocol 4 Pause E Total RNA in Nunc Master Mix twin tec Plate Plate Col 1 2 on Full Screen Gantt Chart Elapsed Time 00 00 00 Silver Insert Reset All Form Selections to Defaults J m BenchCel Information BenchCel Stacker 1 BenchCel Stacker 2 BenchCel Stacker 3 BenchCel Stacker 4 Currently Running Protocol f p Tip Box Empty Empty Empty 5 After verifying that the NGS Workstation has been set up correctly click Run Selected Protocol Run Selected Protocol 24 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation Error messages encountered at start of run After starting the run you may see the error messages displayed below When encountered make the indicated selections and proceed with the run Encountering either or both of these error messages is not indicative of a problem with the NGS workstation or your run setup 1 If you encounter the G axis error message shown below select Ignore and Continue leaving device in cu
9. 476 5 SamplePlatexYZ 01 D1 5 19 6 SamplePlatexYZ E1 E1 5 49 7 SamplePlatexYZ F1 F1 4 86 8 SamplePlatexYZ G1 GI 5 05 9 SamplePlatexYZ H1 H1 4 37 10 SamplePlatexYZ A2 A2 0 11 SamplePlatexYZ 82 B2 0 12 SamplePlatexYZ C2 C2 0 SB Kama nK Ann Danaa ae Et tet nt nnn nn geen A Figure 10 Sample spreadsheet for 100 ng sample aliquot for 1 column run 82 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 You can find a sample spreadsheet in the directory C gt VWorks Workspace gt NGS Option B gt XT_RNA_ILM gt Aliquot Library Input Files gt 100ng_transfer_full_plate_template_xlsx The 100ng_transfer_full_plate_template xlsx file may be copied and used as a template for creating the csv files for each Aliquot_Libraries_v1 0 pro run If you are using the sample file as a template for runs with fewer than 12 columns be sure to retain rows for all 96 wells and populate the Volume column with 0 for unused wells 3 Load the csv file onto the PC containing the VWorks software into a suitable folder such as C gt VWorks Workspace gt NGS Option B gt XT RNA_ILM gt Aliquot Library Input Files 4 Turn on the chiller set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 5 Load the Bravo deck according to Table 40 Table 40 Initial Bravo deck configuration for Aliquot_Libraries_v1 0 pro Location Content
10. 738 pL 984 pL 1353 pL 1968 pL 3690 pL 10 Prepare the appropriate amount of Adaptor Ligation Master Mix containing the SureSelect Ligation Master Mix and the adaptors according to Table 25 below Table 25 Preparation of Adaptor Ligation Master Mix for LibraryPrep_ RNASeq_ILM_v1 1 rst Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2Columns 3Columns 4 Columns 6 Columns 12 Columns Nuclease free water 2 5 pL 30 8 uL 61 5 uL 82 0 uL 112 8 pL 164 0 pL 307 5 pL SureSelect Ligation 5 0 pL 61 5 pL 123 0 uL 164 0 uL 225 5 uL 328 0 uL 615 0 pL Master Mix SureSelect Oligo 5 0 uL 61 5 pL 123 0 pL 164 0 uL 225 5 pL 328 0 uL 615 0 pL Adaptor Mix Total Volume 12 5 pL 153 8 pL 307 5 pL 410 0 pL 563 8 pL 820 0 pL 1537 5 pL 60 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 11 Using the same Nunc DeepWell master mix source plate that was used for the mRNA_Purification_v1 0 pro run prepare the Library Prep master mix source plate Add the volumes indicated in Table 26 of each master mix to all wells of the indicated column of the plate Keep the master mixes on ice during the aliquoting steps The final configuration of the master mix source plate is shown in Figure 6 Table 26 Preparation of the Master Mix Source Plate for LibraryPrep_RNASeq_ILM_v1 1 rst Master Mix Positionon Volume of Master Mix added per Well of Nunc Deep Well S
11. For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 2 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 3 Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 1 uL of each amplified library DNA sample diluted with 3 uL of D1000 sample buffer for the analysis CAUTION Make sure that you thoroughly mix the combined DNA and D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation 4 Load the sample plate or tube strips from step 3 the D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run 5 For each sample measure the concentration of the library ng uL by integrating under the peak at approximately 180 to 550 bp A sample electropherogram is shown in Figure 9 Stopping Point If you do not continue to the next step seal the plate and store at 20 C g 3000 4 Sample Intensity FU 8 MW e bp Figure 9 Analysis of amplified library DNA using the Agilent 2200 TapeStation SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 79 3 Sample Preparation Step 6 Assess library DNA quantit
12. For some samples Bioanalyzer results are improved by diluting 1 uL of the sample in 9 uL of 10 mM Tris 1 mM EDTA prior to analysis Be sure to mix well by vortexing at 2000 rpm on the IKA vortex supplied with the Bioanalyzer before analyzing the diluted samples 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation Verify that the electropherogram shows an average DNA fragment size of approximately 200 to 700 bp A sample electropherogram is shown in Figure 13 If you do not continue to the next step seal the plate and store at 20 C SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 Step 3 Assess DNA quality and quantity 35 100 1 0 300 400 s00 600 1000 2000 10380 Ibp Figure 13 Analysis of indexed DNA using the High Sensitivity DNA Assay SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 121 5 Indexing and Sample Prep for Multiplexed Sequencing CAUTION Option 2 Analysis using the Agilent 2200 TapeStation and High Sensitivity D1000 ScreenTape Use a High Sensitivity D1000 ScreenTape and reagent kit to analyze the indexed DNA For more information to do this step see the Agilent 2200 TapeStation User Manual 1 Seal the DNA sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec Vortex
13. ILM 1 0 pL 12 3 uL 20 5 pL 28 7 uL 36 9 pL 53 3 uL 106 6 pL Reverse PCR Primer Total Volume 33 pL 405 9 pL 676 5 pL 947 1 pL 1217 7 pL 1758 9 pL 3517 8 pL SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 67 3 Sample Preparation 5 Using the same Nunc DeepWell master mix source plate that was used for the LibraryPrep_RNASeq_ILM_v1 1 rst run add the volume of PCR Master Mix indicated in Table 31 to all wells of column 6 of the master mix source plate The final configuration of the master mix source plate is shown in Figure 7 Table 31 Preparation of the Master Mix Source Plate for Pre CapturePCR_RNASeq_ILM_v1 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate n e A Se Sa oe a ae 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs PCR Reaction Mix Column 6 48 6 uL 80 4 uL 114 3 pL 148 1 uL 215 7 pL 435 6 uL A6 H6 If you are using a new DeepWell plate for the pre capture PCR source plate leave columns 1 to 5 empty and add the PCR Master Mix to column 6 of the new plate 68 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 ee seca S A eK ae Figure 7 Configuration of the master mix source plate for Pre CapturePCR_RNASe q_ILM_v1 0 pro Columns 1 5 were used to dispense master mixes during pr
14. Indexing and Sample Prep for Multiplexed Sequencing CAUTION Step 1 Amplify the captured libraries to add index tags In this step the Agilent NGS Workstation completes the liquid handling steps for PCR based addition of indexing tags to the SureSelect enriched DNA samples After the PCR plate is prepared by the Agilent NGS Workstation you transfer the plate to a thermal cycler for amplification To avoid cross contaminating libraries set up PCR master mixes in a dedicated clean area or PCR hood with UV sterilization and positive air flow CAUTION Assign indexes to DNA samples Select the appropriate indexing primer for each sample This guide contains two sets of index sequence information Verify that you are referencing the information appropriate for your kit version before you proceed Kits with indexing primers supplied in a blue plate include 8 bp indexes A01 through H12 See page 133 through page 134 for indexing primer A0Q1 H12 plate map and nucleotide sequence information Kits with indexing primers supplied in a clear plate include 8 bp indexes 1 through 96 See page 139 through page 145 for indexing primer 1 96 plate map and nucleotide sequence information 108 Use a different index primer for each sample to be sequenced in the same lane The number of samples that may be combined per lane depends on the sequencing platform performance and the Capture Library size See Table 57 for sequence data requireme
15. Labware MiniHub according to Table 37 using the plate orientations shown in Figure 4 Table 37 Initial MiniHub configuration for AMPureXP_v1 1 pro Pre Capture PCR Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Empty Empty DeepWell plate Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Eppendorf Empty Empty twin tec Plate Shelf 2 Empty Nuclease free AMPure XP beads Empty water reservoir in Nunc DeepWell from step 7 plate from step 6 Shelf 1 Bottom Empty 70 ethanol Empty Empty tip box reservoir from step 8 10 Load the Bravo deck according to Table 38 Table 38 Initial Bravo deck configuration for AMPureXP_v1 1 pro Pre Capture PCR Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 9 Amplified cDNA samples in PCR plate unsealed and seated on red insert PCR plate type must be specified on setup form under step 2 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 75 Sample Preparation 11 Load the BenchCel Microplate Handling Workstation according to Table 39 Table 39 Initial BenchCel configuration for AMPureXP_v1 1 pro Pre Capture PCR No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty
16. Library For runs that use a single capture library for all rows of the plate prepare the master mix as described in Step a Table 42 or Table 43 below For runs that use different capture libraries for individual rows prepare each master mix as described in Step b Table 44 or Table 45 below 86 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 a For runs that use a single capture library for all rows prepare the SureSelect Capture Library Master Mix as listed in Table 42 or Table 43 based on the Mb target size of your design Table 42 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb 8 rows of wells Target size lt 3 0 Mb SureSelect Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for Reagent 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 4 5 uL 76 5 pL 114 8 uL 153 0 pL 191 3 pL 306 0 uL 612 0 pL water RNase Block 0 5 pL 8 5 uL 12 8 uL 17 0 pL 21 3 uL 34 0 uL 68 0 uL purple cap SureSelect 2 0 pL 34 0 pL 51 0 pL 68 0 pL 85 0 pL 136 0 pL 272 0 uL Capture Library Total Volume 7 0 pL 119 0 pL 178 6 pL 238 0 pL 297 6 pL 476 0 pL 852 0 pL Table 43 Preparation of Capture Library Master Mix for target sizes gt 3 0 Mb 8 rows of wells Target size gt 3 0 Mb SureSelect Volume for Volume for Volume for Volumefor Volumefor Volumefor Volume for Reagent 1 Library 1 Column 2Columns 3Columns 4Columns
17. Pause and Diagnose Continue ie The remainder of the SureSelectCapture amp Wash_v1 0 rst runset takes approximately 1 5 hours Once the runset is complete the captured bead bound DNA samples are located in the Eppendorf plate at position 9 of the Bravo deck When the runset is complete seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec and store the plate on ice while setting up the next automation protocol Captured DNA is retained on the streptavidin beads during the post capture amplification step SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 105 4 Hybridization Step 3 Capture the hybridized DNA 106 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing SureSelect Automated Strand Specific RNA Target Enrichment Protocol 5 Indexing and Sample Prep for Multiplexed Sequencing Step 1 Amplify the captured libraries to add index tags 108 Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads 116 Step 3 Assess DNA quality and quantity 120 Step 4 Pool samples for multiplexed sequencing 124 Step 5 Prepare and analyze sequencing samples 126 This chapter describes the steps to add index tags by amplification then to purify and assess quality and quantity of the libraries in order to pool indexed samples for multiplexed sequencing aa Agilent Technologies 107 5
18. Pipetman P10 P20 P200 P1000 or equivalent SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 15 1 Before You Begin Required Equipment 16 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing SureSelect Automated Strand Specific RNA Target Enrichment Protocol ee 2 7 e Using the Agilent NGS Workstation for SureSelect RNA Library Preparation About the Agilent NGS Workstation 18 Overview of the SureSelect RNA Library Prep Procedure 28 Experimental Setup Considerations for Automated Runs 30 This chapter contains an orientation to the Agilent NGS Workstation an overview of the SureSelect single stranded RNA library preparation and target enrichment protocol and considerations for designing SureSelect RNA experiments for automated processing using the Agilent NGS Workstation wit Agilent Technologies 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation About the Agilent NGS Workstation About the Bravo Platform CAUTION The Bravo platform is a versatile liquid handler with a nine plate location platform deck suitable for handling 96 well 384 well and 1536 well plates The Bravo platform is controlled by the VWorks Automation Control software Fitted with a choice of seven interchangeable fixed tip or disposable tip pipette heads it accurately dispenses fluids from 0 1 uL to 250 uL Before you beg
19. Purify first strand cDNA using AMPure XP beads Synthesize second strand cDNA Repair DNA ends e Purify end repaired DNA Adenylate DNA 3 ends e Ligate adaptors e Purify adaptor ligated DNA Amplify adaptor ligated cDNA library Purify library amplicons using AMPure XP beads Aliquot 100 ng of prepped libraries for hybridization Hybridize prepped DNA to Capture Library Capture and wash DNA hybrids Add index tags by PCR Purify indexed amplicons using AMPure XP beads VWorks Protocols Used for Agilent NGS Workstation automation mRNA_Purification_v1 0 pro AMPureXP_v1 1 pro First Strand LibraryPrep_RNASeq_ILM_v1 1 rst Pre CapturePCR_RNASeq_ILM_v1 0 pro AMPureXP_v1 1 pro Pre Capture PCR Aliquot_Libraries_v1 0 pro SureSelectHybridization_v1 0 pro SureSelectCapture amp Wash_v1 0 rst Post CapturePCR_RNASeq_ILM_v1 0 pro AMPureXP_v1 1 pro Post Capture PCR SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 29 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation Experimental Setup Considerations for Automated Runs Agilent SureSelect Automated Strand Specific RNA Library Prep runs may include 1 2 3 4 6 or 12 columns equivalent to 8 16 24 32 48 or 96 wells of RNA samples to be prepared for sequencing on the Illumina platform Plan your experiments using complete columns of samples Table6 Columns to Samples Equivalency Number of Columns Processe
20. and DNA 1000 Assay Use a Bioanalyzer DNA 1000 chip and reagent kit For more information to do this step see the Agilent DNA 1000 Kit Guide 1 Set up the 2100 Bioanalyzer as instructed in the reagent kit guide 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis 5 Load the prepared chip into the 2100 Bioanalyzer and start the run within five minutes after preparation 6 Measure the concentration of the library ng uL by integrating under the peak at approximately 180 to 550 bp For accurate quantification make sure that the concentration falls within the linear range of the assay A sample electropherogram is shown in Figure 8 If you do not continue to the next step seal the plate and store at 20 C 15 s 100 1 200 30 400 s0 1000 1500 Ibp Figure 8 Analysis of amplified library DNA using a DNA 1000 assay SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Option 2 Analysis using the Agilent 2200 TapeStation and D1000 ScreenTape Use a D1000 ScreenTape and associated reagent kit to analyze the amplified libraries
21. at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 2 Leave tip boxes on shelves 1 and 2 in casette 1 of the Labware MiniHub from the previous LibraryPrep_RNASeq_v1 0 rst run Otherwise clear the remaining positions of the MiniHub and BenchCel of plates and tip boxes 3 Pre set the temperature of Bravo deck position 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks Bravo deck position 6 corresponds to CPAC 2 position 2 on the Multi TEC control touchscreen SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Prepare the PCR reaction mix and the master mix source plate 4 Prepare the appropriate volume of PCR Reaction Mix according to Table 30 Mix well using a vortex mixer and keep on ice Table 30 Preparation of PCR Reaction Mix SureSelect Volumefor Volume for Volume for Volume for Volume for Volume for Volume for Reagent 1 Library 1 Column 2 Columns 3 Columns 4Columns 6 Columns 12 Columns Nuclease free 5 0 uL 61 5 uL 102 5 uL 143 5 uL 184 5 uL 266 5 uL 533 0 uL water RNA Seq PCR 25 0 pL 307 5 pL 512 5 pL 717 5 pL 922 5 pL 1332 5 pL 2665 pL Master Mix Uracil DNA 1 0 pL 12 3 uL 20 5 uL 28 7 uL 36 9 uL 53 3 uL 106 6 pL Glycosylase UDG SureSelect Primer 1 0 pL 12 3 uL 20 5 uL 28 7 uL 36 9 uL 53 3 uL 106 6 pL Forward primer RNA Seq
22. for Illumina Sequencing Before You Begin 1 Safety Notes CAUTION e Wear appropriate personal protective equipment PPE when working in the laboratory SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 11 1 Before You Begin CAUTION Required Reagents Table 1 Required Reagents for SureSelect RNA Target Enrichment Automation Description SureSelect RNA Capture Library SureSelect RNA Reagent Kit Illumina platforms ILM 96 Samples Actinomycin pi DMSO Dynabeads M 270 Streptavidin Beads Agencourt AMPure XP Kit 5 mL 60 mL 450 mL 1X Low TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA 100 Ethanol molecular biology grade Vendor and part number Select one library from Table 2 Agilent p n G9692B Sigma p n A1410 Sigma p n D8418 Life Technologies p n 65306 Beckman Coulter Genomics p n A63880 p n A63881 p n A63882 Life Technologies p n 12090 015 or equivalent Sigma Aldrich p n E7023 3 columns of samples per run Each 96 reaction kit contains sufficient reagents for 96 reactions used in runs that include at least t Actinomycin D should be obtained as a solid and prepared at 4 ug ul concentration in DMSO no more than one month before use See page 35 for more information Use only the recommended Dynabeads M 270 Streptavidin Beads for this automated protocol Use of other streptavidin bead preparations may adversely affect performance and
23. for Multiplexed Sequencing Load the Agilent NGS Workstation 10 Load the Labware MiniHub according to Table 60 using the plate orientations shown in Figure 4 Table 60 Initial MiniHub configuration for Post CapturePCR_RNASeq_ILM_v1 0 pro Vertical Cassette 1 Cassette 2 Cassette 3 Cassette 4 Shelf Position Shelf 5 Empty Empty Empty Empty Top Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Empty Empty Shelf 2 Empty tip box Empty Empty Empty Shelf 1 New tip box Empty Empty Empty tip box Bottom 11 Load the Bravo deck according to Table 61 Table 61 Initial Bravo deck configuration for Post CapturePCR_RNASeq_ILM_v1 0 pro Location Content 4 Captured DNA bead suspensions in Eppendorf twin tec plate 6 Indexing primers in PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 9 Master mix plate containing PCR Master Mix in Column 4 Nunc DeepWell plate seated on silver insert 112 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 12 Load the BenchCel Microplate Handling Workstation according to Table 62 Table 62 Initial BenchCel configuration for Post CapturePCR_RNASeq_ILM_v1 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 1 Tip box Empty Empty Empty 4 1 Tip box Empty Empty Empty 6 1 Tip bo
24. ng sample plate from the Bravo deck and use a vacuum concentrator to dry the sample at lt 45 C 12 Reconstitute each dried sample with 3 4 uL of nuclease free water to bring the final concentration to 29 4 ng uL Pipette up and down along the sides of each well for optimal recovery 13 Seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 14 Vortex the plate for 30 seconds to ensure complete reconstitution then centrifuge the plate for 1 minute to drive the well contents off the walls and plate seal 84 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization Step 2 Hybridize the DNA library and SureSelect RNA Capture Library In this step the Agilent NGS Workstation completes the liquid handling steps in preparation for hybridization of the prepared cDNA samples to one or more SureSelect capture libraries Afterward you transfer the sample plate to a thermal cycler held at 65 C to allow hybridization of the DNA sample to the SureSelect capture library Prepare the workstation 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a NucleoClean decontamination wipe 3 Turn on the ThermoCube and set to 25 C for position 9 of the Bravo deck 4 Place the silver Nunc DeepWell plate insert on position 9 of the Bravo deck This insert is requ
25. plates and tip boxes Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a Nucleoclean decontamination wipe Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time Mix the bead suspension well so that the reagent appears homogeneous and consistent in color Prepare a Nunc DeepWell source plate containing AMPure XP beads For each well to be processed add 95 uL of homogeneous AMPure XP beads per well to the Nunc DeepWell plate Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly prepared 70 ethanol 116 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 8 Load the Labware MiniHub according to Table 64 using the plate orientations shown in Figure 4 Table 64 Initial MiniHub configuration for AMPureXP_v1 1 pro Post Capture PCR Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Empty Empty DeepWell plate Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Eppendorf Empty Empty twin tec plate Shelf 2 Empty Nuclease free AMPure XP beads Empty water reservoir in Nunc DeepWell from step 6 plate from step 5 Shelf 1 Bottom Empty 70 ethanol Empty Empty tip box reservoir from step 7 9 Load the Bravo d
26. term storage is located at position 4 of the Bravo deck 19 When you see the following prompt remove the PCR plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 seconds F Plate ready to seal Seal PCR plate and run thermocyder protocol f User data entry Pause and Diagnose Continue 20 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate air bubbles 114 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 21 Transfer the PCR plate to a thermal cycler and run the PCR amplification program shown in Table 63 Table 63 Post Capture PCR cycling program Segment Number of Temperature Time Cycles 1 1 95 C 2 minutes 2 12 95 C 30 seconds 57 C 30 seconds 72 C 1 minute 3 1 72 C 5 minutes 4 1 4 C Hold SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 5 115 5 Indexing and Sample Prep for Multiplexed Sequencing Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads In this step the Agilent NGS Workstation transfers AMPure XP beads to the indexed DNA sample plate and then collects and washes the bead bound DNA Prepare the workstation and reagents 1 2 Clear the Labware MiniHub and BenchCel of all
27. transfer by the Bravo liquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run Load the Agilent NGS Workstation 14 Load the Labware MiniHub according to Table 27 using the plate orientations shown in Figure 4 Table 27 Initial MiniHub configuration for LibraryPrep_ RNASeq_ILM_v1 1 rst Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Empty Nunc Empty Nunc Empty DeepWell plate DeepWell plate Shelf 4 Empty Empty Eppendorf Empty Eppendorf Empty twin tec plate twin tec plate Shelf 3 Empty Empty Empty Eppendorf Empty twin tec plate Shelf 2 Waste tip box Nuclease free AMPure XP beads Empty retained from water reservoir in Nunc DeepWell Shelf 1 Bottom mRNA Purification protocol Clean tip box retained from mRNA Purification protocol from step 7 70 ethanol reservoir from step 8 plate from step 6 Empty Empty tip box SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 63 3 64 Sample Preparation 15 Load the Bravo deck according to Table 28 Table 28 Initial Bravo deck configuration for LibraryPrep_RNASeq_ILM_v1 1 rst Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 6 Empty Eppendorf twin tec plate oriented with well A1 in the upper left 7 Purified first strand cDNA samples in Eppendorf twin tec plate oriented with well A1 in t
28. transfer the RNA sample plate to position 4 of the Bravo deck seated in the red insert Carefully unseal the plate then click Continue Post RNA Elution Once the incubation is complete get plate from thermal cycler and place at position 4 Carefully remove the seal When finished dick Continue below 27 The workstation adds RNA Seq Bead Binding Buffer to the eluted RNA samples and then holds the samples at room temperature for 5 minutes to allow the poly A RNA to re bind the beads 46 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 28 When the workstation has finished the collecting and washing the bound RNA samples in this second round of purification you will be prompted by VWorks as shown below Remove and discard the PCR plate from position 4 then click Continue Remove Plate Remove the plate from position 4 When finished dick Continue below SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 47 3 48 Sample Preparation 29 The workstation adds RNA Seq Fragmentation Mix to the bead bound RNA samples in preparation for the RNA fragmentation step When the workstation has finished you will be prompted by VWorks as shown below e RNA Fragmentation Get plate from position 6 seal at 165 C for 1 0s Place in thermal cyder using the following program 94 C for 8 minu
29. verify the simulation mode status in VWorks using the following steps 1 Verify that Simulation is off is displayed on the status indicator accessible by clicking View gt Control Toolbar 2 Ifthe indicator displays Simulation is on click the status indicator button to turn off the simulation mode a Diagnostics 3 S Log out H Compile gt Start EE Pause a If you cannot see the toolbar above the SureSelect VWorks form click the Full Screen button to exit full screen mode If the toolbar is still not visible right click on the form and then select Control Toolbar from the menu Finishing a protocol or runset The window below appears when each run is complete Click Yes to release the BenchCel racks to allow removal of components used in the current run in preparation for the next pro or rst run i Protocol complete u 2 Release stacker racks used in protocols mm ce K SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 27 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation Overview of the SureSelect RNA Library Prep Procedure Figure 2 summarizes the SureSelect workflow for RNA samples to be sequenced using the Illumina sequencing platform For each sample to be sequenced an individual cDNA library is prepared The libraries are then target enriched and tagged by PCR with an index sequence Depending on the capacity of the sequencin
30. 1 4 pL 28 9 pL 36 3 pL 58 6 pL 118 1 pL Table 45 Preparation of Capture Library Master Mix for target sizes gt 3 0 Mb single row of wells Target size gt 3 0 Mb SureSelect Volume for Volume for Volume for Volumefor Volumefor Volume for Volume for Reagent 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 1 5 pL 3 0 uL 4 6 uL 6 2 uL 7 8 uL 12 6 uL 25 3 uL water RNase Block 0 5 pL 1 0 pL 1 5 pL 2 1 uL 2 6 uL 4 2 uL 8 4 uL purple cap SureSelect 5 0 uL 10 0 pL 15 3 pL 20 6 pL 25 9 uL 41 9 uL 84 4 uL Capture Library Total Volume 7 0 pL 14 0 pL 21 4 pL 28 9 pL 36 3 pL 58 6 pL 118 1 pL 88 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 Prepare the Hybridization Buffer master mix 7 Prepare the appropriate volume of Hybridization Buffer Master Mix at room temperature as indicated in Table 46 Table 46 Preparation of Hybridization Buffer Master Mix SureSelect Volume for Volume for Volume for Volume for Volume for Volume for Reagent 1 Column 2 Columns 3 Columns 4 Columns 6 Columns 12 Columns SureSelect Hyb 1 140 9 pL 197 3 pL 250 0 pL 310 1 pL 422 8 uL 789 3 uL SureSelect Hyb 2 5 6 pL 7 9 pL 10 0 pL 12 4 uL 16 9 uL 31 6 uL red cap SureSelect Hyb 3 56 4 uL 78 9 uL 100 0 pL 124 0 uL 169 1 pL 315 7 uL yellow cap SureSelect Hyb 4 73 3 pL 102 6 pL 130 0 pL 161 2 pL 219 9 pL 410 4 uL Total Volume 276 2 pL 386 7 pL 490 0 pL 607 7 pL 828 7 pL 15
31. 227 7202 3 Rights in Commercial Computer Software or Com puter Software Documentation Notice to Purchaser Limited License Use of this product is covered by one or more of the following US patents and corre sponding patent claims outside the US 6 258 569 6 171 785 6 127 155 6 030 787 5 994 056 5 876 930 5 804 375 5 789 224 5 773 258 claims 1 and 6 only 5 723 591 5 677 152 claims 1 to 23 only 5 618 711 5 538 848 and claims outside the US corre sponding to expired US Patent No 5 079 352 The purchase of this product includes a limited non transferable immu nity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim and no right to perform commercial services of any kind including without limitation report ing the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Fur ther information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lin coln Centre Drive Foster City California 94404 USA 2 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Safety Notices CAUTION A CAUTION notice denotes a hazard It calls atten
32. 3 4 6 or 12 columns 20 Click Display Initial Workstation Setup Display Initial Workstation Setup 21 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup 4 MiniHub t MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 mind Shelf 5 g daa ISRA ee a P A a ae eee AT oe a SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 22 When verification is complete click Run Selected Protocol Run Selected Protocol 23 When ready to begin the run click OK in the following window If the temperature of Bravo deck position 4 was not pre set to 66 C the runset will pause while position 4 reaches temperature VWorks This runset contains protocols that will start running as soon as P possible Before you click OK verify that the system is ready for the runs to start If you are not ready to start a run immediately click Cancel cone SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 103 4 Hybridization CAUTION It is important to complete step 24 quickly and carefully Transfer the sample plate to the Bravo platform quickly to retain the 65 C sample temperature Unseal the plate without tilting or jerking the plate to avoid sample splashing Make sure that the Agilent NGS Workstation is completely prepared with dec
33. 33 48 Index Number Sequence 33 CACTTCGA 34 CAGCGTTA 35 CATACCAA 36 CCAGTTCA 37 CCGAAGTA 38 CCGTGAGA 39 CCTCCTGA 40 CGAACTTA 41 CGACTGGA 42 CGCATACA 43 CTCAATGA 44 CTGAGCCA 45 CTGGCATA 46 GAATCTGA 47 GACTAGTA 48 GAGCTGAA 142 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Table 85 SureSelect RNA Seq Indexes 49 64 Index Number Sequence 49 GATAGACA 50 GCCACATA 51 GCGAGTAA 52 GCTAACGA 53 GCTCGGTA 54 GGAGAACA 55 GGTGCGAA 56 GTACGCAA 57 GTCGTAGA 58 GTCTGTCA 59 GTGTTCTA 60 TAGGATGA 61 TATCAGCA 62 TCCGTCTA 63 TCTTCACA 64 TGAAGAGA SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 143 6 Reference Table 86 SureSelect RNA Seq Indexes 65 80 Index Number Sequence 65 TGGAACAA 66 TGGCTTCA 67 TGGTGGTA 68 TTCACGCA 69 AACTCACC 70 AAGAGATC 71 AAGGACAC 72 AATCCGTC 73 AATGTTGC 74 ACACGACC 75 ACAGATTC 76 AGATGTAC 77 AGCACCTC 78 AGCCATGC 79 AGGCTAAC 80 ATAGCGAC 144 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Table 87 SureSelect RNA Seq Indexes 81 96 Index Number Sequence 81 ATCATTCC 82 ATTGGCTC 83 CAAGGAGC 84 CACCTTAC 85 CCATCCTC 86 CCGACAAC 87 CCTAATCC 88 CCTCTATC 89 CGACACAC 90 CGGATTGC 91 CTAAGGTC 92 GAACAGGC 93 GACAGTGC 94 GAGTTAGC 95 GATGAATC 96 GCCAAGAC SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Seque
34. 47 pL 8 If precipitate forms warm the hybridization buffer at 65 C for 5 minutes SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 89 4 Hybridization Prepare the master mix source plate 9 Ina Nunc DeepWell plate prepare the master mix source plate containing the master mixes prepared in step 5 to step 7 at room temperature Add the volumes indicated in Table 47 of each master mix to each well of the indicated column of the Nunc DeepWell plate When using multiple capture libraries in a run add each Capture Library Master Mix to the appropriate row s of the Nunc DeepWell plate The final configuration of the master mix source plate is shown in Figure 11 Table 47 Preparation of the Master Mix Source Plate for SureSelectHybridization_v1 0 pro Master Mix Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate Solution Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Block Master Mix Column 1 17 0 uL 29 4 uL 41 7 uL 54 0 uL 78 7 uL 158 8 pL A1 H1 Capture Library Column 2 14 0 pL 21 4 uL 28 9 uL 36 3 pL 51 2 uL 99 5 uL Master Mix A2 H2 Hybridization Column 3 30 5 uL 44 3 uL 57 2 uL 71 9 uL 99 5 uL 189 3 pL Buffer Master Mix A3 H3 90 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 tees lt lt lt amp 3eo ee Ce A Figure 11 Configura
35. 6 Columns 12 Columns Nuclease free 1 5 pL 25 5 uL 38 3 uL 51 0 uL 63 8 uL 102 0 pL 204 0 pL water RNase Block 0 5 pL 8 5 uL 12 8 uL 17 0 pL 21 3 uL 34 0 uL 68 0 uL purple cap SureSelect 5 0 uL 85 0 pL 127 5 uL 170 0 pL 212 5 pL 340 0 uL 680 0 pL Capture Library Total Volume 7 0 pL 119 0 pL 178 6 pL 238 0 pL 297 6 pL 476 0 pL 852 0 pL SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 87 4 Hybridization Table 44 Preparation of Capture Library Master Mix for target sizes lt 3 0 Mb single row of wells b For runs that use different capture libraries in individual rows prepare a SureSelect Capture Library Master Mix for each capture library as listed in Table 44 or Table 45 based on the Mb target size of your design The volumes listed in Table 44 and Table 45 are for a single row of sample wells If a given capture library will be hybridized in multiple rows multiply each of the values below by the number of rows assigned to that capture library Target size lt 3 0 Mb SureSelect Volume for Volume for Volume for Volumefor Volumefor Volumefor Volume for Reagent 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns Nuclease free 4 5 uL 9 0 uL 13 8 uL 18 6 uL 23 3 uL 37 7 uL 75 9 uL water RNase Block 0 5 pL 1 0 pL 1 5 pL 2 1 uL 2 6 uL 4 2 uL 8 4 uL purple cap SureSelect 2 0 pL 4 0 uL 6 1 pL 8 3 pL 10 4 pL 16 8 uL 33 8 uL Capture Library Total Volume 7 0 pL 14 0 pL 2
36. A Library Prep and Target Enrichment for Illumina Sequencing 137 6 Reference Table 80 SureSelect Target Enrichment Box 2 Content Kit Component Details SureSelect Hyb 3 tube with yellow cap SureSelect Indexing Block 1 tube with green cap SureSelect Block 2 tube with blue cap SureSelect ILM Indexing Block 3 tube with brown cap SureSelect RNase Block tube with purple cap 138 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 Table 81 Plate map for RNA Seq Indexes 1 96 8 bp provided in clear plate in Library Prep kit p n 5500 0117 1 2 3 4 5 6 7 8 9 10 11 12 A Index Index Index Index Index Index Index Index Index Index Index Index 1 9 17 25 33 41 49 57 65 73 81 89 B Index Index Index Index Index Index Index Index Index Index Index Index 2 10 18 26 34 42 50 58 66 74 82 90 C Index Index Index Index Index Index Index Index Index Index Index Index 3 11 19 27 35 43 51 59 67 75 83 91 D Index Index Index Index Index Index Index Index Index Index Index Index 4 12 20 28 36 44 52 60 68 76 84 92 E Index Index Index Index Index Index Index Index Index Index Index Index 5 13 21 29 37 45 53 61 69 77 85 93 F Index Index Index Index Index Index Index Index Index Index Index Index 6 14 22 30 38 46 54 62 70 78 86 94 G Index Index Index Index Index Index Index Index Index Index Index Index 7 15 23 31 39 47 55 63 71 79 87 95 H Index Index Index Index Index Index Index Index Index
37. Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 1 1 1 1 1 1 96 Eppendorf twin tec full skirt 1 1 1 1 1 1 plates SureSelectCapture amp Wash_v1 0 rst Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 2 3 4 6 11 Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 2 2 2 2 2 2 96 Eppendorf twin tec full skirt 2 2 2 2 2 2 plates Thermo Scientific Reservoir 1 1 1 1 1 1 Axygen square well plate waste 1 1 1 1 1 1 148 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 Post CapturePCR_RNASeq_ILM_v1 0 pro Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 2 2 2 2 2 2 Empty tip boxes for waste tips 2 2 2 2 2 2 PCR plates compatible with 1 1 1 1 1 1 thermal cycler AMPureXP_v1 1 pro Post Capture PCR Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 2 2 3 6 Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 2 2 2 2 2 2 96 Eppendorf twin tec full skirt 1 1 1 1 1 1 plates Thermo Scientific Reservoirs 2 2 2 2 2 2 Axygen square well plate waste 1 1 1 1 1 1 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 149 www agilent com In This Book This guide contains information t
38. Index Index Index 8 16 24 32 40 48 56 64 72 80 88 96 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 139 6 Reference Nucleotide Sequences of SureSelect RNA Seq Indexes Original Kit Configuration The nucleotide sequence of each SureSelect RNA Seq Index provided in the original kit configuration is provided in the tables below Refer to the sequence information below only if your kit includes Library Prep kit p n 5500 0117 with indexing primers provided in a clear 96 well plate Each index is 8 nt in length See page 126 for sequencing run setup requirements for sequencing libraries using 8 bp indexes Table 82 SureSelect RNA Seq Indexes 1 16 Index Number Sequence 1 AACGTGAT 2 AAACATCG 3 ATGCCTAA 4 AGTGGTCA 5 ACCACTGT 6 ACATTGGC 7 CAGATCTG 8 CATCAAGT 9 CGCTGATC 10 ACAAGCTA 11 CTGTAGCC 12 AGTACAAG 13 AACAACCA 14 AACCGAGA 15 AACGCTTA 16 AAGACGGA 140 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Table 83 SureSelect RNA Seq Indexes 17 32 Index Number Sequence 17 AAGGTACA 18 ACACAGAA 19 ACAGCAGA 20 ACCTCCAA 21 ACGCTCGA 22 ACGTATCA 23 ACTATGCA 24 AGAGTCAA 25 AGATCGCA 26 AGCAGGAA 27 AGTCACTA 28 ATCCTGTA 29 ATTGAGGA 30 CAACCACA 31 CAAGACTA 32 CAATGGAA SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 141 6 Reference Table 84 SureSelect RNA Seq Indexes
39. R plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck positions 4 and 6 The plate type selected must be compatible with the thermal cycler to be used for incubation steps during the protocol During run setup be sure to use the plate type selected from this menu at positions 4 and 6 of the Bravo deck In addition when the workstation issues prompts to add plates to postion 4 or 6 during the run use only the same PCR plate type specified here 18 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 3 Select Number of Columns of Samples o Display Initial We ding to Workstatior 19 Click Display Initial Workstation Setup Display Initial Workstation Setup 20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup a MiniHub t MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 mini Shelf 5 J BIA Gamo eel aene e ae ae 40 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 21 When verification is complete click Run Selected Protocol Run Selected Protocol If workstation devices do not respond when you start the run but activity is recorded in the Log verify that VWorks is not running in Simulation mode See page 27 for more information Running the mRNA_Purificat
40. RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 If the final volume of the combined index tagged samples is less than the desired final volume V f add Low TE to bring the volume to the desired level If the final volume of the combined index tagged samples is greater than the final desired volume V f lyophilize and reconstitute to the desired volume 3 If you store the library before sequencing add Tween 20 to 0 1 v v and store at 20 C short term Exact library pool dilution and processing can vary based on the flow cell capacity and analysis pipeline versions being used Refer to the appropriate Illumina user guide for instructions SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 125 5 Indexing and Sample Prep for Multiplexed Sequencing Step 5 Prepare and analyze sequencing samples Proceed to cluster amplification using the Illumina Paired End Cluster Generation Kit refer to the manufacturer s instructions for this step The optimal seeding concentration for cluster amplification from SureSelect RNA target enriched libraries is approximately 8 10 pM The optimal seeding concentration may vary depending on the method used for library quantification and fragment size distribution This protocol has been validated with 2 x 100 base paired end reads However read length can be adjusted to achieve the desired res
41. Sequence ACGTATCA GTCTGTCA CTAAGGTC D07 CGACACAC E07 CCGTGAGA F07 GTGTTCTA G07 CAATGGAA H07 AGCACCTC A08 CAGCGTTA B08 TAGGATGA C08 AGTGGTCA C11 CGACTGGA D08 ACAGCAGA D11 CAAGACTA E08 CATACCAA E11 CCTCCTGA Sequence AACTCACC GCTAACGA CAGATCTG Sequence ATGCCTAA GAATCTGA AACGTGAT D01 CACTTCGA D04 ATCCTGTA E01 GCCAAGAC E04 CTGTAGCC F01 GACTAGTA F04 GCTCGGTA G01 ATTGGCTC G04 ACACGACC H01 GATGAATC H04 AGTCACTA A02 AGCAGGAA A05 AACGCTTA B02 GAGCTGAA B05 GGAGAACA c02 AAACATCG c05 CATCAAGT D02 GAGTTAGC D05 AAGGTACA E02 CGAACTTA E05 CGCTGATC F02 GATAGACA F05 GGTGCGAA G02 AAGGACAC G05 CCTAATCC H02 GACAGTGC H05 CTGAGCCA A03 ATCATTCC A06 AGCCATGC B03 GCCACATA B06 GTACGCAA C03 ACCACTGT c06 AGTACAAG D03 CTGGCATA D06 ACATTGGC E03 ACCTCCAA E06 ATTGAGGA F03 GCGAGTAA F06 GTCGTAGA G03 ACTATGCA G06 AGAGTCAA CGGATTGC CCGACAAC F08 TATCAGCA F11 TGGTGGTA G08 ATAGCGAC G11 AACAACCA H08 ACGCTCGA H11 AATCCGTC A09 CTCAATGA Al2CAAGGAGC B09 TCCGTCTA B12 TTCACGCA C09 AGGCTAAC C12 CACCTTAC D09 CCATCCTC D12 AAGACGGA E09 AGATGTAC E12 ACACAGAA F12 GAACAGGC G12 AACCGAGA ACAAGCTA F09 TCTTCACA G09 CCGAAGTA CGCATACA 134 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 Reference Informa
42. Strand End SureSelect Strand Specific RNA Library Prep page 60 Repair Enzyme Mix Kit ILM Box 1 20 C RNA Seq Second Strand End SureSelect Strand Specific RNA Library Prep page 60 Repair Oligo Mix Kit ILM Box 1 20 C SureSelect Ligation Master Mix SureSelect Strand Specific RNA Library Prep page 60 Kit ILM Box 1 20 C SureSelect Oligo Adaptor Mix SureSelect Strand Specific RNA Library Prep page 60 Kit ILM Box 1 20 C RNA Seq dA Tailing Master Mix SureSelect Strand Specific RNA Library Prep page 61 Kit ILM Box 1 20 C SureSelect Strand Specific RNA Library Prep master mixes are viscous Mix thoroughly by vortexing before removing an aliquot for use and after combining the master mixes with other solutions 58 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Prepare the workstation 1 Turn on the ThermoCube set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 2 Leave tip boxes on shelves 1 and 2 in casette 1 of the Labware MiniHub from the previous mRNA_Purification_v1 0 pro run Otherwise clear the remaining MiniHub and BenchCel positions of plates and tip boxes 3 Pre set the temperature of Bravo deck position 4 to 14 C and of position 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks On t
43. SureSelect Automated RNA Target Enrichment Strand Specific RNA Library Prep and Target Enrichment for Illumina Pai Version C 0 Decembe SureSelect platform manufactur with Agilent SurePrint Technology Research Use Only Not for use in Diagnostic Procedures cae Agilent Technologies Notices Agilent Technologies Inc 2014 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 69691 90020 Edition Version C 0 December 2014 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Acknowledgement Oligonucleotide sequences 2006 2008 and 2011 Illumina Inc All rights reserved Only for use with the Illumina sequencer systems and associated assays Technical Support For technical product support contact your local Agilent Support Services representa tive For US and Canada call 800 227 9770 option 3 4 4 For other countries find your support center telephone numbers at www agilent com chem contactus Or send an e mail to SureSelect Support agilent com Notice to Purchaser Research Use Only Not for use in diagnostic procedures Warranty The material contained in this document is
44. Time Step 1 65 C 5 minutes Step 2 4 C 1 minute Step 3 20 C 5 minutes Step 4 20 C Hold 42 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Step 1 Purify poly A RNA fragment RNA synthesize first strand cDNA 23 After the thermal cycler reaches the 20 C Hold step and when prompted by the dialog below transfer the RNA sample plate to position 4 of the Bravo deck seated in the red insert Carefully unseal the plate then click Continue Post RNA Denaturation Once the incubation is complete get plate from thermal cycler and place at position 4 Carefully remove the seal When finished click Continue below SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 43 3 Sample Preparation 24 When the workstation has finished collecting and washing the bead bound RNA samples you will be prompted by VWorks as shown below i Replace Plate Remove the plate from position 4 Replace the plate at position 4 with a new plate Ensure that the same plate type is used When finished dick Continue below a Remove and discard the PCR plate from position 4 of the Bravo deck b Place a fresh PCR plate at position 4 seated in the red insert The PCR plate type added here must be the same plate type as the one removed and as was specified during the run setup c After positioning the plate clic
45. ample Prep for Multiplexed Sequencing Step 4 Pool samples for multiplexed sequencing The number of indexed libraries that may be multiplexed in a single sequencing lane is determined by the output specifications of the platform used together with the amount of sequencing data required for your research design Calculate the number of indexes that can be combined per lane according to the capacity of your platform and the amount of sequencing data required per sample 1 Combine the libraries such that each index tagged sample is present in equimolar amounts in the pool For each library use the formula below to determine the amount of indexed sample to use Volume of Index Vis xC x C i where V f is the final desired volume of the pool C f is the desired final concentration of all the DNA in the pool is the number of indexes and C i is the initial concentration of each indexed sample Table 67 shows an example of the amount of 4 index tagged samples of different concentrations and Low TE needed for a final volume of 20 uL at 10 nM Table 67 Example of indexed sample volume calculation for total volume of 20 uL Component V f C i C f Volume to use pL Sample 1 20 uL 20 nM 10 nM 4 2 5 Sample 2 20 uL 10 nM 10 nM 4 5 Sample 3 20 uL 17 nM 10 nM 4 2 9 Sample 4 20 uL 25 nM 10 nM 4 2 Low TE 76 2 Adjust the final volume of the pooled library to the desired final concentration 124 SureSelect Automated
46. at this stage Each hybridization reaction will contain 100 ng of the appropriate prepped cDNA sample Before starting the hybridization step you must create a table containing instructions for the Agilent NGS Workstation indicating the volume of each sample required for a 100 ng aliquot 1 Create a csv comma separated value file with the headers shown in Figure 10 The header text must not contain spaces The table may be created using a spreadsheet application such as Microsoft Excel software and then saved in csv format The file must include rows for all 96 wells of the plate 2 Enter the information requested in the header for each DNA sample e In the SourceBC field enter the sample plate description or barcode The SourceBC field contents must be identical for all rows e In the SourceWell and DestinationWell fields enter each well position for the plate SourceWell and DestinationWell field contents must be identical for a given sample In the Volume field enter the volume in uL equivalent to 100 ng DNA for each sample These values are determined from the concentration values obtained from Bioanalyzer or TapeStation traces in the previous section For all empty wells on the plate enter the value 0 as shown in Figure 10 do not delete rows for empty wells A B C D 1 SourceBC SowrceWell DestinationWell Volume 2 SamplePlatexYZ Al Al 5 35 3 SamplePlateXYZ 81 B1 4 28 4 SamplePlatexYZ C1 C1
47. c reservoir containing 45 mL of freshly prepared 70 ethanol 54 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 9 Load the Labware MiniHub according to Table 20 using the plate orientations shown in Figure 4 Table 20 Initial MiniHub configuration for AMPureXP_v1 1 pro First Strand Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Empty Empty DeepWell plate Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Eppendorf Empty Empty twin tec Plate Shelf 2 Waste tip box Nuclease free AMPure XP beads Empty retained from water reservoir in Nunc DeepWell mRNA Purification from step 7 plate from step 6 protocol Shelf 1 Bottom Clean tip box 70 ethanol Empty Empty tip box retained from reservoir from mRNA Purification step 8 protocol The tip boxes retained in Cassette 1 are not shown on the VWorks Workstation Setup table These tip boxes are not used in AMPureXP_v1 1 pro First Strand but are used in a later protocol This labware should be retained in the MiniHub to ensure that empty and full tip positions are properly defined for the subsequent protocol 10 Load the Bravo deck according to Table 21 Table 21 Initial Bravo deck configuration for AMPureXP_v1 1 pro First Strand Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 9 First strand cDNA samples in PCR plate seated o
48. cientific reservoirs place the notched corner facing the center of the hub SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 14 Load the Bravo deck according to Table 11 Table 11 Initial Bravo deck configuration for mRNA_Purification_v1 0 pro Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 4 Oligo dT beads in PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 6 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 7 Total RNA samples in twin tec plate 9 Master Mix Source Plate seated on silver insert Nunc DeepWell see Figure 3 on page 37 for column content 15 Load the BenchCel Microplate Handling Workstation according to Table 12 Table 12 Initial BenchCel configuration for mRNA_Purification_v1 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 2 Tip boxes Empty Empty Empty 3 3 Tip boxes Empty Empty Empty 4 3 Tip boxes Empty Empty Empty 6 5 Tip boxes Empty Empty Empty 12 9 Tip boxes Empty Empty Empty SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 39 3 Sample Preparation Run VWorks protocol mRNA_Purification_v1 0 pro 16 On the SureSelect setup form under Select Protocol to Run select mRNA_Purification_v1 0 pro 17 Under Select PC
49. cing Indexing and Sample Prep for Multiplexed Sequencing 5 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini Shelf 5 Minne EE SNA U amai i T a ae a a 16 When verification is complete click Run Selected Protocol Run Selected Protocol The purification protocol takes approximately 45 minutes When complete the purified DNA samples are in the Eppendorf plate located on Bravo deck position 7 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 119 5 Indexing and Sample Prep for Multiplexed Sequencing Step 3 Assess DNA quality and quantity Stopping Point 120 Option 1 Analysis using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Assay 1 Set up the 2100 Bioanalyzer as instructed in the High Sensitivity DNA Assay kit guide Version B 02 07 or higher of the Agilent 2100 Expert Software is required for High Sensitivity DNA Assay Kit runs 2 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 3 Vortex the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal 4 Prepare the chip samples and ladder as instructed in the reagent kit guide using 1 uL of each sample for the analysis
50. ct SureSelectHybridization_v1 0 pro 15 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate containing the DNA samples at position 6 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 16 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 17 Click Display Initial Workstation Setup Display Initial Workstation Setup 18 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup 4 MiniHub 1 MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 min Shelf 5 J A ShA nO a AT aa a T ante ett BPs Pewee 19 When verification is complete click Run Selected Protocol Run Selected Protocol The Agilent NGS Workstation transfers SureSelect Block Master Mix to the prepped DNA containing wells of the PCR plate When this process is complete you will be prompted to transfer the plate to the thermal cycler for sample denaturation prior to hybridization SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 93 4 Hybridization 20 When prompted by VWorks as shown below remove the PCR plate from position 6 of the Bravo deck leaving the red insert in place r Remove plate Remove plate from insert seal and place in thermocyder User data entry Pause and Diagnose Con
51. ct Ligation Master Mix tube with purple cap SureSelect Oligo Adaptor Mix tube with blue cap RNA Seq PCR Master Mix bottle Uracil DNA Glycosylase UDG tube with yellow cap SureSelect Primer tube with brown cap RNA Seq ILM Reverse PCR Primer tube with black cap RNA Seq ILM Post capture PCR Primer tube with green cap SureSelect Indexes 8 bp SureSelect 8 bp Indexes A01 through H12 provided in blue 96 well plate See Table 75 on page 134 for index sequences Tt See Table 74 on page 133 for a plate map SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 131 6 Reference Table 71 SureSelect Strand Specific RNA Library Prep ILM Box 2 Content Kit Component Details Oligo dT Microparticles bottle RNA Seq Bead Binding Buffer bottle RNA Seq Bead Washing Buffer bottle RNA Seq Bead Elution Buffer bottle Nuclease Free Water bottle Table 72 SureSelect Target Enrichment Box 1 Content Kit Component Details SureSelect Hyb 1 tube with orange cap SureSelect Hyb 2 tube with red cap SureSelect Hyb 4 tube with black cap SureSelect Binding Buffer bottle SureSelect Wash Buffer 1 bottle SureSelect Wash Buffer 2 bottle SureSelect Elution Buffer bottle SureSelect Neutralization Buffer bottle The provided SureSelect Elution Buffer and Neutralization Buffer are not used in the auto mated RNA Target Enrichment workflow described in this manual 132 SureSelect Automa
52. d Reference Information for Kits with Revised Index Configuration indexing primers in blue plate Use the reference information in this section if your kit includes Library Prep Kit p n 5500 0135 If your kit does not include this component kit see page 135 for kit content and indexing primer information Kit Contents 130 The SureSelect RNA Reagent Kits contain the following component kits Table 69 SureSelect RNA Seq Kit Content Revised Index Configuration Component Kits Storage Condition Part Number SureSelect Strand Specific RNA Library 20 C 5500 0135 Prep ILM Box 1 SureSelect Strand Specific RNA Library 4 C 5190 6411 Prep ILM Box 2 SureSelect Target Enrichment Box 1 Room Temperature 5190 4394 SureSelect Target Enrichment Box 2 20 C 5190 6262 SureSelect capture libraries and reagents must be used within one year of receipt SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 The contents of each of the component kits listed in Table 69 are described in the tables below Table 70 SureSelect Strand Specific RNA Library Prep ILM Box 1 Content Revised Index Configuration Kit Component Details RNA Seq Fragmentation Mix bottle RNA Seq First Strand Master Mix tube with orange cap RNA Seq Second Strand End Repair Enzyme Mix bottle RNA Seq Second Strand End Repair Oligo Mix tube with yellow cap RNA Seq dA Tailing Master Mix bottle SureSele
53. d Total Number of Samples Processed 1 8 2 16 3 24 4 32 6 48 12 96 The number of columns or samples that may be processed using the supplied reagents will depend on the experimental design For greatest efficiency of reagent use plan experiments using at least 3 columns per run Each 96 reaction kit contains sufficient reagents for 96 reactions configured as 4 runs of 3 columns of samples per run 30 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing CAUTION Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Considerations for Placement of RNA Samples in 96 well Plates for Automated Processing e The Agilent NGS Workstation processes samples column wise beginning at column 1 RNA samples should be loaded into 96 well plates column wise in well order Al to H1 then A2 to H2 ending with A12 to H12 When processing partial runs with lt 12 sample columns do not leave empty columns between sample columns always load the plate using the left most column that is available For sample indexing by PCR see Figure 2 you will need to prepare a separate plate containing the indexing primers Assign the wells to be indexed with their respective indexing primers during experimental design This guide includes information for kits containing two different sets of indexing primers Verify that you are referencing the information appropriate for your kit version before you proc
54. dd 5 uL of the appropriate indexing primer to the appropriate wells of a PCR plate The well position for each index should correspond to the position of the RNA sample assigned to that index in the original total RNA sample plate Keep the plate on ice 6 Prepare the appropriate volume of PCR master mix according to Table 58 Mix well using a vortex mixer and keep on ice Table 58 Preparation of PCR Master Mix for Post CapturePCR_RNASeq_ILM_v1 0 pro SureSelect Volume for Volume for Volume for Volumefor Volumefor Volume for Volume for Reagent 1 Library 1 Column 2 Columns 3Columns 4Columns 6 Columns 12 Columns RNA Seq PCR 25 0 uL 307 5 uL 512 5 pL 717 5 uL 922 5 uL 1332 5 uL 2665 pL Master Mix RNA Seq ILM 1 0 pL 12 3 pL 20 5 pL 28 7 pL 36 9 pL 53 3 uL 106 6 pL Post Capture PCR Primer Total Volume 26 0 pL 319 8 pL 533 0 pL 746 2 pL 959 4 pL 1385 8 pL 2771 6 pL SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 109 5 Indexing and Sample Prep for Multiplexed Sequencing 7 Using the same Nunc DeepWell master mix source plate that was used for the SureSelectHybridization_v1 0 pro protocol add the volume of PCR master mix indicated in Table 59 to all wells of column 4 of the master mix source plate The final configuration of the master mix source plate is shown in Figure 12 Table 59 Preparation of the Master Mix Source Plate for Post CapturePCR_RNASeq_ILM_v1 0 pro Master Mix Solution
55. designations Bravo Deck Position Designation on Inheco Multi TEC Control Screen 4 CPAC 21 6 CPAC 22 1 Using the arrow buttons select the appropriate block CPAC 2 block 1 or CPAC 2 block 2 crac 2 lt gt 24 9 C Shaker 0200 rpm SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 19 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 To set the temperature of the selected block press the SET button CPAC 2 1 24 9 C 2 Shaker 0200 rpm 3 Using the numeral pad enter the desired temperature The entered temperature appears in the top left rectangle Once the correct temperature is displayed press the rectangle to enter the temperature 4 Press the Temp button until the new temperature is displayed on the SET button and until the Temp button is darkened indicating that the selected heat block is heating or cooling to the new temperature setting The current temperature of the block is indicated in the center of the display CPAC 2 1 25 0 C E Current temp 0200 rpm se 20 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Setting the Temperature of Bravo Deck Position 9 Using the ThermoCube Device Bravo deck position 9 is equipped with a ThermoCube thermoelectric temperature control system used
56. e vious protocols 6 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 7 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles The presence of bubbles in source plate solutions may cause inaccurate volume transfer by the Bravo liquid handling platform Ensure that the source plate is sealed and centrifuged prior to use in a run SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 69 3 70 Sample Preparation Load the Agilent NGS Workstation 8 Load the Labware MiniHub according to Table 32 using the plate orientations shown in Figure 4 Table 32 Initial MiniHub configuration for Pre CapturePCR_RNASeq_ILM_v1 0 pro Vertical Cassette 1 Cassette 2 Cassette 3 Cassette 4 Shelf Position Shelf 5 Empty Empty Empty Empty Top Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Empty Empty Empty Shelf 2 Waste tip box Empty Empty Empty retained from Library Prep protocol Shelf 1 Clean tip box Empty Empty Empty tip box Bottom retained from Library Prep protocol 9 Load the Bravo deck according to Table 33 Table 33 Initial Bravo deck configuration for Pre CapturePCR_RNASeq_ILM_v1 0 pro Location Content 6 Empty PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 7 Prepped cDNA samples in Eppendorf tw
57. e net to calculate RNA sequencing metrics it is important to include the parameter STRAND_SPECIFICITY SECOND_READ_TRANSCRIPTION_STRAND to correctly calculate the strand specificity metrics SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 127 5 Indexing and Sample Prep for Multiplexed Sequencing Step 5 Prepare and analyze sequencing samples 128 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing SureSelect Automated Strand Specific RNA Target Enrichment Protocol 6 Reference Reference Information for Kits with Revised Index Configuration indexing primers in blue plate 130 Reference Information for Kits with Original Index Configuration indexing primers in clear plate 135 Plasticware quantities for automation protocols 146 This chapter contains reference information including component kit contents index sequences and plasticware requirements ot Agilent Technologies 129 6 Reference CAUTION This chapter contains two sets of index sequence and kit content information The first section covers kits with indexing primers supplied in Library Prep Kit p n 5500 0135 typically received December 2014 or later The second section covers kits with indexing primers supplied in Library Prep Kit 5500 0117 typically received before December 2014 Verify that you are referencing the information appropriate for your kit version before you procee
58. e prompted to transfer the plate I User data entry SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 95 4 Hybridization 24 When prompted by VWorks as shown below quickly remove the sample plate from the thermal cycler unseal the plate carefully to avoid splashing and transfer the plate to position 6 of the Bravo deck seated in the red insert Click Continue Place DNA plate on Bravo Complete the following steps as quickly as possible Retrieve DNA plate from thermocycler and place on carrier at Bravo position 6 and l unseal i Click Continue to resume protocol Use Caution Position 6 will be hot User data entry Pause and Diagnose Continue WARNING Bravo deck position 6 will be hot Use caution when handling components that contact heated deck positions The Agilent NGS Workstation transfers the capture library hybridization buffer mixture to the wells of the PCR plate containing the mixture of prepped DNA samples and blocking agents 96 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 25 When prompted by VWorks as shown below quickly remove the PCR plate from Bravo deck position 6 leaving the insert in place Remove Plate from 6 Quickly remove plate from position 6 seal and place in thermocycler Click Continue after plate is in thermocycler for protocol to fini
59. earch goals Sequencing run setup guidelines for 8 bp indexes Sequencing runs must be set up to perform an 8 nt index read For the HiSeq platform use the Cycles settings shown in Table 68 Cycle number settings can be specified on the Run Configuration screen of the instrument control software interface after choosing Custom from the index type selection buttons For complete index sequence information see the Reference section starting on page 129 Table 68 HiSeq platform Run Configuration screen Cycle Number settings Run Segment Cycle Number Read 1 100 Index 1 i7 9 Index 2 i5 0 Read 2 100 Settings apply to v3 0 SBS chemistry 126 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 Sequence analysis guidelines The SureSelect RNA sequencing library preparation method preserves RNA strandedness as described here The first strand of cDNA is the reverse complement of the poly A RNA transcript strand Since the second strand of cDNA is eliminated before PCR the sequence of read 1 which starts at the P5 end matches only the first strand of cDNA Read 2 which starts at the P7 end matches the second strand of cDNA the poly A RNA transcript strand When running analysis of this data to determine strandedness it is important to include this information For example when using the Picard tools http picard sourceforg
60. eck according to Table 65 Table 65 Initial Bravo deck configuration for AMPureXP_v1 1 pro Post Capture PCR Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 9 Indexed library samples in PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 117 5 Indexing and Sample Prep for Multiplexed Sequencing 10 Load the BenchCel Microplate Handling Workstation according to Table 66 Table 66 Initial BenchCel configuration for AMPureXP_v1 1 pro Post Capture PCR No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty Run VWorks protocol AMPureXP_v1 1 pro Post Capture PCR 11 On the SureSelect setup form under Select Protocol to Run select AMPureXP_v1 1 pro Post Capture PCR 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate containing the DNA samples at position 9 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Workstation Setup Display Initial Workstation Setup 118 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequen
61. eed Kits with indexing primers supplied in a blue plate include 8 bp indexes A01 through H12 See page 133 through page 134 for indexing primer A01 H12 plate map and nucleotide sequence information Kits with indexing primers supplied in a clear plate include 8 bp indexes 1 through 96 See page 139 through page 145 for indexing primer 1 96 plate map and nucleotide sequence information Protocol steps for indexing using primers provided in either configuration are identical Considerations for Equipment Setup e Some workflow steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer Several workflow steps require that the sample plate be sealed using the PlateLoc thermal microplate sealer on the Agilent NGS Workstation and then centrifuged to collect any dispersed liquid To maximize efficiency locate the centrifuge in close proximity to the Agilent NGS Workstation SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 31 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation PCR Plate Type Considerations Automation protocols include several liquid handling steps in which reagents are dispensed to PCR plates in preparation for transfer to a thermal cycler For these steps you must specify the PCR plate type to be
62. en verification is complete click Run Selected Protocol Run Selected Protocol Running the AMPureXP_v1 1 pro First Strand protocol takes approximately 45 minutes During this time you can prepare the purification reagents for the Library Prep automation protocol as described on page 59 Once the AMPureXP_v1 1 pro First Strand protocol is complete the purified cDNA samples are located in the Eppendorf plate at position 7 of the Bravo deck Proceed immediately to Step 3 Prepare cDNA libraries for Hybridization on page 58 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 3 57 3 Sample Preparation CAUTION Step 3 Prepare cDNA libraries for Hybridization This step is automated using the LibraryPrep_RNASeq_ILM_v1 1 rst runset During the runset the Agilent NGS Workstation completes second strand cDNA library synthesis and end modification steps including end repair A tailing and adaptor ligation After certain modification steps the Agilent NGS Workstation purifies the prepared cDNA using AMPure XP beads This step uses the SureSelect RNA Reagent Kit components listed in Table 23 in addition to the purification reagents prepared for use on page 59 Thaw each reagent vial and keep on ice Vortex each vial for 5 seconds to mix before use Table 23 Reagents for automation runset LibraryPrep_RNASeq_ILM_v1 1 rst Kit Component Storage Location Where Used RNA Seq Second
63. ents 135 Nucleotide Sequences of SureSelect RNA Seq Indexes Original Kit Configuration 140 Plasticware quantities for automation protocols 146 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina SureSelect Automated Strand Specific RNA Target Enrichment Protocol ee 1 070 Before You Begin s Procedural Notes 10 Safety Notes 11 Required Reagents 12 Required Equipment 14 Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment This protocol describes automated sample processing using the Agilent NGS Workstation For non automated sample processing procedures for Agilent s SureSelect Strand Specific RNA Target Enrichment Kit for sequencing on the Illumina platform see publication G9691 90000 ot Agilent Technologies 9 1 10 Before You Begin Procedural Notes Certain protocol steps require the rapid transfer of sample plates between the Bravo deck and a thermal cycler Locate your thermal cycler in close proximity to the Agilent NGS Workstation to allow rapid and efficient plate transfer Use of Agilent s SureCycler 8800 thermal cycler and associated plasticware is recommended for optimal performance The workflow is compatible with additional thermal cyclers but performance should be validated before running a large number of samples See page 32 fora list of supported PCR pla
64. fresh 120 ng uL Actinomycin D dilution in water from a stock solution of 4 ug uL Actinomycin D in DMSO according to Table 16 Table 16 Preparation of 120 ng ul Actinomycin D Reagent Volume for up to 12 column run includes excess Actinomycin D 4 ug ul in DMSO 3 pL Nuclease free water 97 pL Total 100 pL SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 49 3 Sample Preparation CAUTION c Prepare the appropriate amount of RNA Seq First Strand Master Mix Actinomycin D mixture on ice according to the table below SureSelect Strand Specific RNA Library Prep master mixes are viscous Mix thoroughly by vortexing before removing an aliquot for use and after combining the master mixes with other solutions Table 17 Preparation of First Strand Master Mix Actinomycin D mixture Reagent RNA Seq First Strand Master Mix Actinomycin D 120 ng pl in H20 Total Volume Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns 8 0 uL 98 4 uL 196 8 uL 262 4 uL 360 8 uL 492 0 uL 918 4 uL 0 5 uL 6 2 uL 12 3 uL 16 4 uL 22 6 uL 30 8 uL 57 4 uL 8 5 pL 104 6 pL 209 1 pL 278 8 pL 383 4 pL 522 8 pL 975 8 pL d Add the volume listed in Table 18 of the First Strand Master Mix Actinomycin D mixture to column 2 of the Master Mix source plate at position 9 of the Bravo deck The final configuration of the sou
65. g platform up to 96 samples can be pooled and sequenced in a single lane using the multiplex index tags that are provided with the SureSelect Strand Specific RNA Library Prep kit Total RNA samples 1 2 n SureSelect X7 RNA Paves RNA NGS Target Enrichment poly A RNA Workflow Fragment RNA using RNA Seq Fragmentation Mix RNA fragments Prepare samples using SureSelect Strand Specife R RNA Library Prep Kit esign target 7 z sequences in eArray Adaptor ligated cDNA library iE PCR amplify Suet RNA Capture Library Prepared cDNA library amplicons Hybridize using SureSelect reagents and protocol Capture hybrids on magnetic beads PCR amplify using RNA Seq Index primer Pool libraries for multiplex sequencing Optional i 1 1 v Figure 2 Overall sequencing sample preparation workflow 28 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Table 5 summarizes how the VWorks protocols are integrated into the Strand Specific RNA Library Prep and Target Enrichment workflow See the Sample Preparation chapter for complete instructions for use of the VWorks protocols for sample processing Table 5 Overview of VWorks protocols and runsets used during the workflow Workflow Step Purify poly A RNA using oligo dT beads e Chemically fragment the poly A RNA Synthesize first strand cDNA
66. he control touchscreen Bravo deck positions 4 corresponds to CPAC 2 position 1 while deck position 6 corresponds to CPAC 2 position 2 Prepare the purification reagents 4 Verify that the AMPure XP bead suspension is at room temperature Do not freeze the beads at any time 5 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 6 Prepare a Nunc DeepWell source plate for the beads by adding 160 uL of homogeneous AMPure XP beads per well for each well to be processed 7 Prepare a Thermo Scientific reservoir containing 20 mL of nuclease free water 8 Prepare a separate Thermo Scientific reservoir containing 150 mL of freshly prepared 70 ethanol SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 59 3 Sample Preparation Prepare the master mix source plate 9 Prepare the appropriate amount of RNA Seq Second Strand End Repair Master Mix according to Table 24 below Table 24 Preparation of RNA Seq Second Strand End Repair Master Mix for LibraryPrep_ RNASeq_ILM_v1 1 rst Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns RNA Seq Second 25 0 uL 307 5 uL 615 pL 820 uL 1127 5 uL 1640 uL 3075 pL Strand End Repair Enzyme Mix RNA Seq Second 5 0 uL 61 5 pL 123 pL 164 pL 225 5 uL 328 uL 615 uL Strand End Repair Oligo Mix Total Volume 30 pL 369 pL
67. he upper left 9 Library Prep Master Mix Source Plate Nunc DeepWell unsealed and seated on silver insert 16 Load the BenchCel Microplate Handling Workstation according to Table 29 Table 29 Initial BenchCel configuration for LibraryPrep_ RNASeq_ILM_v1 1 rst No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 2 Tip boxes Empty Empty Empty 2 3 Tip boxes Empty Empty Empty 3 4 Tip boxes Empty Empty Empty 4 5 Tip boxes Empty Empty Empty 6 7 Tip boxes Empty Empty Empty 12 11 Tip boxes 3 Tip boxes Empty Empty Run VWorks runset LibraryPrep_RNASeq_ILM_v1 1 rst 17 On the SureSelect setup form under Select Protocol to Run select LibraryPrep_RNASeq_ILM_v1 1 rst 18 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 19 Click Display Initial Workstation Setup Display Initial Workstation Setup 20 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup MiniHub MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini ae Ae Shelf 5 7 ren A ENA A a Ea e een aAA ee 21 When verification is complete click Run Selected Protocol Run Selected Protocol 22 When ready to begin the run click OK in the following window E VWorks G This runset contains
68. in make sure that you have read and understand operating maintenance and safety instructions for using your Bravo platform Refer to the Bravo Platform User Guide G5409 90006 and the VWorks Software User Guide G5415 90063 18 Bravo Platform Deck The protocols in the following sections include instructions for placing plates and reagent reservoirs on specific Bravo deck locations Use Figure 1 to familiarize yourself with the location numbering convention on the Bravo platform deck Back I E 1 T a 4 5 6 E T 8 9 Front Figure 1 Bravo platform deck SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Setting the Temperature of Bravo Deck Heat Blocks Bravo deck positions 4 and 6 are equipped with Inheco heat blocks used to incubate sample plates at defined temperatures during the run Runs that include high 85 C or low 4 C temperature incubation steps may be expedited by pre setting the temperature of the affected block before starting the run Bravo deck heat block temperatures may be changed using the Inheco Multi TEC Control device touchscreen as described in the steps below See Table 4 for designations of the heat block containing Bravo deck positions on the Multi TEC control device Table 4 Inheco Multi TEC Control touchscreen
69. in 25 uL of nuclease free water For optimal performance total RNA samples should have an RNA Integrity Number RIN of 8 or more based on analysis using Agilent s 2100 Bioanalyzer A workstation operator must be present during this automation protocol to transfer plates between the workstation which completes most liquid handling steps and the thermal cycler which is used for several incubation steps In addition the operator must prepare and dispense a master mix immediately before it is used in the automation protocol see step 30 on page 49 Prepare the workstation 1 Open the SureSelect setup form using the SureSelect_RNA_ILM VWForm shortcut on your desktop 2 Log in to the VWorks software 3 Turn on the ThermoCube set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 4 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 34 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing CAUTION Sample Preparation 3 Prepare reagents for the run 5 Bring the reagents listed in Table 8 to room temperature Table 8 Reagents for poly A RNA purification and RNA fragmentation Kit Component Storage Location Where Used in Protocol Oligo dT Microparticles RNA Library Prep Kit Box 2 4 C page 36 RNA Seq Bead Washing Buffer RNA Library Prep Kit Box 2 4 C page 36 RNA Seq Bead Elution Buffer RNA Library Prep K
70. in tec plate oriented with well A1 in the upper left 9 Master mix source plate Nunc DeepWell unsealed and seated on silver insert SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 10 Load the BenchCel Microplate Handling Workstation according to Table 34 Table 34 Initial BenchCel configuration for Pre CapturePCR_RNASeq_ILM_v1 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 1 Tip box Empty Empty Empty 4 1 Tip box Empty Empty Empty 6 1 Tip box Empty Empty Empty 12 1 Tip box Empty Empty Empty Run VWorks protocol Pre CapturePCR_RNASeq_ILM_v1 0 pro 11 On the SureSelect setup form under Select Protocol to Run select Pre CapturePCR_RNASeq_ILM_v1 0 pro 12 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate that was loaded on Bravo deck position 6 The plate type selected must be compatible with the thermal cycler to be used for amplification 13 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 14 Click Display Initial Workstation Setup O Display Initial Workstation Setup 15 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup a MiniHub t MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini Shelf 5
71. ion_v1 0 pro protocol takes approximately 90 minutes including four incubation periods on the thermal cycler During the automation protocol run a workstation operator must be present to transfer plates between the workstation and thermal cycler when prompted as detailed on the following pages SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 41 3 Sample Preparation 22 When the workstation has finished combining the RNA samples with the oligo dT beads you will be prompted by VWorks as shown below r RNA Denaturation Get plate from position 4 seal at 165 C for 1 0s Place in thermal cycler using the following program 65 C for 5 minutes 4 C for 1 minute l I 20 C for 5 minutes 20 C Hold When finished dick Continue below i User data entry Pause and Diagnose Continue C a Remove the plate from position 4 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 seconds b Briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid without pelleting the beads c Transfer the PCR plate to a thermal cycler with the heated lid ON and run the RNA denaturation bead binding program shown in Table 13 After transferring the plate click Continue on the VWorks screen Table 13 Thermal cycler program for RNA denaturation and RNA bead binding Step Temperature
72. ired to facilitate heat transfer to DeepWell source plate wells during the Hybridization protocol SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 4 85 4 Hybridization Prepare the SureSelect Block master mix 5 Prepare the appropriate volume of SureSelect Block master mix on ice as indicated in Table 41 Table 41 Preparation of SureSelect Block Master Mix SureSelect Volume for Volume for Volume for Volume for Volume for Volume for Volume for Reagent 1 Library 1 Column 2 Columns 3 Columns 4Columns 6 Columns 12 Columns Nuclease free 6 0 uL 76 5 pL 127 5 pL 178 5 pL 229 5 uL 331 5 uL 663 0 uL water SureSelect 2 5 uL 31 9 uL 53 1 pL 74 4 uL 95 6 uL 138 1 pL 276 3 pL Indexing Block 1 green cap SureSelect Block 2 5 uL 31 9 pL 53 1 pL 74 4 uL 95 6 uL 138 1 pL 276 3 pL 2 blue cap SureSelect 0 6 pL 77 yL 12 8 pL 17 9 pL 23 0 pL 33 2 uL 66 3 uL Indexing Block 3 brown cap Total Volume 11 6 pL 147 9 pL 246 5 pL 345 1 pL 443 7 pL 640 9 pL 1281 9 pL Prepare one or more SureSelect Capture Library master mixes 6 Prepare the appropriate volume of SureSelect capture library master mix for each of the capture libraries that will be used for hybridization as indicated in Table 42 to Table 45 Mix the components by pipetting Keep the master mixes on ice during preparation and aliquoting Each row of the prepped cDNA sample plate may be hybridized to a different SureSelect Capture
73. is not supported by Agilent 12 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Before You Begin 1 Table 2 SureSelect RNA Capture Libraries Capture Library 96 Samples Custom RNA Capture 1 kb up to 499 kb 5190 7281 reorder 5190 7282 Custom RNA Capture 0 5 Mb up to 2 9 Mb 5190 7283 reorder 5190 7284 Custom RNA Capture 3 Mb up to 5 9 Mb 5190 7285 reorder 5190 7286 SureSelect RNA Kinome 5190 7287 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 13 Before You Begin Required Equipment Table3 Required Equipment Description Agilent NGS Workstation Option B with VWorks software version 11 3 0 1195 Bravo 96 well PCR plate insert red Robotic Pipetting Tips Sterile Filtered 250 uL Thermal cycler and accessories PCR plates compatible with selected Thermal Cycler e g Agilent semi skirted PCR plate for the SureCycler 8800 Thermal Cycler See page 32 for a list of supported PCR plates for automation protocols Eppendorf twin tec full skirted 96 well PCR plates Thermo Scientific Reservoirs Nunc DeepWell Plates sterile 1 3 mL well volume Axygen 96 Deep Well Plate 2 2 mL Square Well waste reservoirs NucleoClean Decontamination Wipes Vacuum concentrator Magnetic separator Vendor and part number Contact Agilent Automation Solutions for ordering information Customerservice automation agilent com
74. it Box 2 4 C page 36 RNA Seq Bead Binding Buffer RNA Library Prep Kit Box 2 4 C page 36 RNA Seq Fragmentation Mix RNA Library Prep Kit Box 1 20 C page 37 6 Locate or prepare a stock solution of 4 ug uL Actinomycin D in DMSO A 3 uL aliquot of this DMSO stock solution will be used on page 49 to prepare a fresh dilution of 120 ng uL Actinomycin D in water for the run The 4 ug uL Actinomycin D in DMSO stock solution must be prepared less than one month prior to use and stored in aliquots at 20 C protected from light To ensure strand specificity you must prepare the 120 ng uL Actinomycin D dilution immediately before use on page 49 Prepare the RNA samples source plate 7 Place 25 uL of each RNA sample 0 2 4 ug RNA in nuclease free water into the wells of a 96 well Eppendorf twin tec plate Load samples into the plate column wise in well order A1 to H1 then A2 to H2 ending with A12 to H12 for processing on the Agilent NGS Workstation SureSelect Strand Specific RNA Library Prep runs may include 1 2 3 4 6 or 12 columns of the plate See Using the Agilent NGS Workstation for SureSelect RNA Library Preparation for additional sample placement considerations SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 35 3 Sample Preparation Prepare the oligo dT beads and mRNA purification source plates When preparing each of the source plates below add the indicated amount of reagent
75. k Continue on the VWorks screen 44 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 25 When the workstation has finished collecting and washing the bead bound RNA samples you will be prompted to transfer the plate to the thermal cycler for the RNA Elution step as shown below F RNA Elution Get plate from position 4 seal at 165 C for 1 0s Place in thermal cyder using the following program 80 C for 2 minutes l 4 C for 1 minute 4 C Hold When finished click Continue below User data entry Pause and Diagnose Continue Ci a Remove the plate from position 4 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 seconds b Briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid without pelleting the beads c Transfer the PCR plate to a thermal cycler with the heated lid ON and run the RNA elution program shown in Table 14 After transferring the plate click Continue on the VWorks screen Table 14 Thermal cycler program for RNA elution Step Temperature Time Step 1 80 C 2 minutes Step 2 4 C 1 minute Step 3 4 C Hold SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 45 3 Sample Preparation 26 After the thermal cycler reaches the 4 C Hold step and when prompted by the dialog below
76. k platforms at temperature and all components in place before you transfer the sample plate to the Bravo deck 24 When prompted by VWorks as shown below quickly remove the PCR plate containing the hybridization reactions held at 65 C from the thermal cycler Unseal the plate carefully to avoid splashing and quickly transfer the plate to position 4 of the Bravo deck seated in red insert Click Continue to resume the runset Add Hyb Plate Complete the following steps as quickly as possible Retrieve Hybridization plate from thermocyder and place on Red insert at i Bravo position 4 and unseal Click Continue to resume protocol Use Caution Position 4 will be hot User data entry Pause and Diagnose Continue WARNING Bravo deck position 4 will be hot Use caution when handling components that contact heated deck positions 104 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 25 When the hybridization samples have been transferred from the PCR plate to the capture plate wells you will be prompted by VWorks as shown below Remove the PCR plate from position 4 of the Bravo deck leaving the red insert in place When finished click Continue to resume the runset ii Update Bravo Deck Remove PCR plate from position 4 Leave Red Aluminum PCR plate insert at position 4 for next protocol r il User data entry
77. lect RNA Library Prep Procedure 28 Experimental Setup Considerations for Automated Runs 30 Considerations for Placement of RNA Samples in 96 well Plates for Automated Processing 31 Considerations for Equipment Setup 31 PCR Plate Type Considerations 32 Sample Preparation 33 Step 1 Purify poly A RNA fragment RNA synthesize first strand cDNA 34 Step 2 Purify first strand cDNA using AMPure XP beads 54 Step 3 Prepare cDNA libraries for Hybridization 58 Step 4 Amplify cDNA libraries by PCR 66 Step 5 Purify amplified DNA using AMPure XP beads 74 Step 6 Assess library DNA quantity and quality 78 Hybridization 81 Step 1 Aliquot prepped DNA libraries for hybridization 82 Step 2 Hybridize the DNA library and SureSelect RNA Capture Library 85 Step 3 Capture the hybridized DNA 98 Contents Indexing and Sample Prep for Multiplexed Sequencing 107 Step 1 Amplify the captured libraries to add index tags 108 Step 2 Purify the amplified indexed libraries using Agencourt AMPure XP beads 116 Step 3 Assess DNA quality and quantity 120 Step 4 Pool samples for multiplexed sequencing 124 Step 5 Prepare and analyze sequencing samples 126 Reference 129 Reference Information for Kits with Revised Index Configuration indexing primers in blue plate 130 Kit Contents 130 Nucleotide Sequences of SureSelect Indexes A01 toH12 134 Reference Information for Kits with Original Index Configuration indexing primers in white plate 135 Kit Cont
78. me of RNA Seq Fragmentation Mix see Table 9 to all wells of Column 1 of a Nunc DeepWell plate The configuration of the source plate is shown in Figure 3 Table9 Preparation of the Master Mix Source Plate for mRNA _Purification_v1 0 pro Master Mix Solution Position on Volume of Master Mix added per Well of Nunc Deep Well Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs RNA Seq Fragmentation Column 1 28 5 uL 47 5 uL 66 5 pL 85 5 pL 123 5 uL 247 0 uL Mix A1 H1 Figure 3 Initial configuration of master mix source plate for mRNA_Purification_v1 0 pro SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 37 3 38 Sample Preparation Load the Agilent NGS Workstation 13 Load the Labware MiniHub according to Table 10 using the plate orientations shown in Figure 4 Table 10 Initial MiniHub configuration for mRNA_Purification_v1 0 pro Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Nunc Empty Nunc Empty Nunc Empty DeepWell plate DeepWell plate DeepWell plate Shelf 4 Empty Bead Binding Buffer Empty Empty in twin tec plate Shelf 3 Empty Bead Elution Buffer Empty Empty in twin tec plate Shelf 2 Empty tip box Empty Bead Wash Buffer in Empty Nunc DeepWell plate Shelf 1 Bottom New tip box Empty Empty Empty tip box Figure 4 Agilent Labware MiniHub plate orientation For Thermo S
79. n red insert PCR plate type must be specified on setup form under step 2 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 55 3 Sample Preparation 11 Load the BenchCel Microplate Handling Workstation according to Table 22 Table 22 Initial BenchCel configuration for AMPureXP_v1 1 pro First Strand No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 6 Tip boxes Empty Empty Empty Run VWorks protocol AMPureXP_v1 1 pro First Strand 12 On the SureSelect setup form under Select Protocol to Run select AMPureXP_vl1 1 pro First Strand 13 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate containing the cDNA samples at position 9 14 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 15 Click Display Initial Workstation Setup Display Initial Workstation Setup 16 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup a MiniHub MiniHub Cassette 1 MiniHiub Cassette 2 MiniHub Cassette 3 Mini Shelf 5 4 i ShA M E N Oe T T a 56 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 17 Wh
80. ncing Reference 6 145 6 Reference Plasticware quantities for automation protocols The tables below show the quantity of each plasticware type used in each automation protocol in the workflow Quantities listed in the tables only include unique labware that was not used in other protocols or runsets For example Nunc DeepWell master mix plates may be reused in multiple protocols but are counted below only where first used mRNA_Purification_v1 0 pro Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 2 3 4 4 6 10 Empty tip boxes for waste tips 2 2 2 2 2 2 Nunc DeepWell Plates 5 5 5 5 5 5 96 Eppendorf twin tec full skirt 3 3 3 3 3 3 plates PCR plates compatible with 4 4 4 4 4 4 thermal cycler Axygen square well plate waste 1 1 1 1 1 1 AMPureXP_v1 1 pro First Strand Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 2 2 3 6 Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 2 2 2 2 2 2 96 Eppendorf twin tec full skirt 1 1 1 1 1 1 plates Thermo Scientific Reservoirs 2 2 2 2 2 2 Axygen square well plate waste 1 1 1 1 1 1 146 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing LibraryPrep RNASeq_ILM_v1 1 rst Reference 6 Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Run
81. nt guidelines Calculate the number of indexes that can be combined per lane based on these guidelines Table 57 Sequencing data requirement guidelines Capture Library Size Recommended Amount of Sequencing Data per Sample 1 kb up to 499 kb 0 1 to 50 Mb 0 5 Mb up to 2 9 Mb 50 to 290 Mb 3 Mb up to 5 9 Mb 300 to 590 Mb Human RNA Kinome 320 Mb For custom libraries Agilent recommends analyzing 100X amount of sequencing data com pared to the Capture Library size for each sample Pool samples according to your expected sequencing output SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing 5 Prepare the workstation Clear the Labware MiniHub and BenchCel of plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo decks and BenchCel with a Nucleoclean decontamination wipe 3 Turn on the ThermoCube set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol Place the silver insert at position 9 4 Pre set the temperature of Bravo deck positions 4 and 6 to 4 C using the Inheco Multi TEC control touchscreen as described in Setting the Temperature of Bravo Deck Heat Blocks On the control touchscreen Bravo deck positions 4 corresponds to CPAC 2 position 1 while deck position 6 corresponds to CPAC 2 position 2 Prepare the index and PCR Master Mix source plates 5 A
82. o run the SureSelect Automated RNA Target Enrichment protocol using the Agilent NGS Workstation Agilent Technologies Inc 2014 Version C 0 December 2014 G9691 90020 oe Agilent Technologies
83. onsiderations for designing SureSelect experiments for automated processing using the Agilent NGS Workstation Sample Preparation This chapter describes the steps to prepare strand specific mRNA libraries from total RNA samples for sequencing on the Illumina platform Hybridization This chapter describes the steps to hybridize the prepped library with the SureSelect RNA capture library and to capture the targeted sequences for enrichment SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 5 Indexing and Sample Prep for Multiplexed Sequencing This chapter describes the steps to index by amplification purify and assess the quality and quantity of the target enriched samples Indexed samples are pooled by mass prior to sequencing 6 Reference This chapter contains reference information SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing What s New in Version C 0 Support for kits supplied with either of two indexing primer configurations Kits with revised index configuration typically received December 2014 or later include indexing primers A01 through H12 provided in a blue plate For kit content details see page 130 For nucleotide sequences of the 8 bp indexes in this revised configuration see Table 75 on page 134 Kits with original index configuration typically received before December 2014 include indexing primers 1 96 provided in a clea
84. ource Plate Solution Source Plate ae a a a a E 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs RNA Seq Second Column 3 42 4 uL 88 5 uL 119 3 uL 165 4 uL 242 3 uL 457 5 uL Strand End A3 H3 Repair Master Mix from Table 24 RNA Seq dA Tailing Column 4 30 0 uL 50 0 pL 70 0 pL 90 0 pL 130 0 pL 260 0 pL Master Mix A4 H4 Adaptor Ligation Column 5 17 7 uL 36 9 uL 49 7 uL 68 9 uL 100 9 pL 190 6 pL Master Mix from A5 H5 Table 25 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 61 3 Sample Preparation xX 3 gt ea oo 1s O A N eS 44 e Canta Sse Ca oe If you are using a new DeepWell plate for the Library Prep Master Mix source plate leave columns 1 and 2 empty and add the PCR Master Mix to columns 3 to 5 of the new plate Figure 6 Configuration of the master mix source plate for LibraryPrep_RNASeq_ ILM_v1 1 rst 12 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 62 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 13 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix source plate on ice The presence of bubbles in source plate solutions may cause inaccurate volume
85. pare cDNA libraries for Hybridization 58 Step 4 Amplify cDNA libraries by PCR 66 Step 5 Purify amplified DNA using AMPure XP beads 74 Step 6 Assess library DNA quantity and quality 78 This section contains instructions for RNA library preparation specific to the Illumina paired read sequencing platform and to automated processing using the Agilent NGS Workstation For each sample to be sequenced individual library preparations are performed in separate wells of a 96 well plate The samples are then target enriched and indexed by PCR amplification allowing multiplexing of up to 96 samples for sequencing on Illumina platforms Refer to Illumina s protocol Preparing Samples for Paired End Sequencing p n 1005361 or the appropriate Illumina protocol for more information Ee Agilent Technologies 33 3 Sample Preparation Step 1 Purify poly A RNA fragment RNA synthesize first strand cDNA In this step automation protocol mRNA_Purification_v1 0 pro is used to complete multiple steps of the RNA Library Preparation workflow First poly A RNA is purified from total RNA using two serial rounds of binding to oligo dT magnetic particles After purification the poly A RNA is chemically fragmented to the appropriate size and then is converted to first strand cDNA Total RNA samples containing 200 ng to 4 ug RNA are suitable for the mRNA library preparation automation protocol Each total RNA sample must be prepared for the run
86. protocols that will start running as soon as possible Before you click OK verify that the system is ready for the runs to start If you are not ready to start a run immediately click Cancel con Running the LibraryPrep_RNASeq_ILM_v1 1 rst runset takes approximately 3 hours Once complete the purified adaptor ligated cDNA samples are located in the Eppendorf plate at position 7 of the Bravo deck Stopping Point If you do not continue to the next step seal the plate and store at 20 C SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 65 3 3 Sample Preparation CAUTION Step 4 Amplify cDNA libraries by PCR In this step the Agilent NGS Workstation completes the liquid handling steps for PCR amplification of the adaptor ligated cDNA samples After the reactions are set up by the workstation you transfer the PCR plate to a thermal cycler for amplification The amplification cycle number is based on the initial amount of total RNA sample used for library preparation Use the SureSelect Strand Specific RNA Library Prep Kit Box 1 for this step Thaw and mix the reagents listed in Table 30 below and keep on ice SureSelect Strand Specific RNA Library Prep master mixes are viscous Mix thoroughly by vortexing before removing an aliquot for use and after combining the master mixes with other solutions Prepare the workstation 1 Turn on the ThermoCube set to 0 C
87. provided as is and is subject to being changed with out notice in future editions Fur ther to the maximum extent permitted by applicable law Agi lent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchant ability and fitness for a particular purpose Agilent shall not be lia ble for errors or for incidental or consequential damages in con nection with the furnishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc ument that conflict with these terms the warranty terms in the separate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend U S Government Restricted Rights Soft ware and technical data rights granted to the federal government include only those rights customarily provided to end user cus tomers Agilent provides this customary commercial license in Software and techni cal data pursuant to FAR 12 211 Technical Data and 12 212 Computer Software and for the Department of Defense DFARS 252 227 7015 Technical Data Commercial Items and DFARS
88. r each well to be processed add 200 uL of the homogeneous bead suspension to the Nunc DeepWell plate 8 Place the streptavidin bead source plate at position 5 of the Bravo deck Prepare capture and wash solution source plates 9 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water 10 Prepare an Eppendorf twin tec source plate labeled Wash 1 For each well to be processed add 160 uL of SureSelect Wash Buffer 1 11 Prepare a Nunc DeepWell source plate labeled Wash 2 For each well to be processed add 800 uL of SureSelect Wash Buffer 2 12 Place the silver Nunc DeepWell plate insert on position 6 of the Bravo deck This insert is required to facilitate heat transfer to DeepWell source plate wells during the Capture amp Wash runset 13 Place the Wash 2 source plate on the silver insert at position 6 of the Bravo deck Make sure the plate is seated properly on the silver DeepWell insert SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 Load the Agilent NGS Workstation 14 Load the Labware MiniHub according to Table 54 using the plate orientations shown in Figure 4 Table 54 Initial MiniHub configuration for SureSelectCapture amp Wash_v1 0 rst Vertical Shelf Cassette 1 Cassette 2 Cassette 3 Cassette 4 Position Shelf 5 Top Empty Empty Empty Empty Shelf 4 Empty Empty Empty Empty Shelf 3 Empty Eppendorf Empty Wash 1 Empty plate Eppendo
89. r plate For kit content details see page 135 For nucleotide sequences of the 8 bp indexes in this original configuration see Table 82 on page 140 through Table 87 on page 145 What s New in Version B 0 Support for revised kit configuration now including RNA Seq Second Strand End Repair Enzyme Mix and RNA Seq Second Strand End Repair Oligo Mix both replacing RNA Seq Second Strand End Repair Master Mix Protocol modifications to support the revised kit configuration begin on page 58 and end on page 61 See Table 70 on page 131 for updated kit contents Updated DMSO supplier information Table 1 on page 12 Updated ordering information for Agilent 2200 TapeStation ScreenTapes and reagents Table 3 on page 15 Updated total RNA preparation considerations Note on page 34 Updated sequence analysis information including library strandedness page 126 to page 127 Footnote removed from Table 1 on page 12 and Note removed from page 130 6 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Content SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Before You Begin 9 Procedural Notes 10 Safety Notes 11 Required Reagents 12 Required Equipment 14 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 17 About the Agilent NGS Workstation 18 About the Bravo Platform 18 VWorks Automation Control Software 22 Overview of the SureSe
90. rce plate is shown in Figure 6 After adding the master mix to the source plate click Continue on the VWorks screen Table 18 Preparation of the Master Mix Source Plate for mRNA _Purification_v1 0 pro Master Mix Solution Position on Source Plate Volume of Master Mix added per Well of Nunc Deep Well Source Plate 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs RNA Seq First Strand Master Mix Actinomycin D mixture Column 2 A2 H2 12 0 pL 25 1 uL 33 8 uL 46 9 uL 64 3 uL 120 9 uL 50 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 3 Sample Preparation Final configuration of master mix source plate for mRNA_Purification_v1 0 pro Figure 5 51 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 3 Sample Preparation 31 With the RNA sample plate still on the thermal cycler the workstation prepares the remaining components for first strand cDNA synthesis When the workstation has finished you will be prompted by VWorks as shown below r Post RNA Fragmentation Once the incubation is complete get plate from thermal cycler and briefly spin down to drive condensation from the inner walls of the wells to the well bottoms Place the plate at position 4 and carefully remove the seal When finished dick Continue below a After the thermal cycler reaches the 4 C Hold step for
91. rf source plate Shelf 2 Empty Nuclease free Empty Empty water reservoir Shelf 1 Bottom Empty Empty Empty Empty tip box 15 Load the Bravo deck according to Table 55 positions 5 and 6 should already be loaded Table 55 Initial Bravo deck configuration for SureSelectCapture amp Wash_v1 0 rst Location Content 1 Empty waste reservoir Axygen 96 Deep Well Plate square wells 4 Empty red aluminum insert 5 Dynabeads M 270 streptavidin bead DeepWell source plate 6 Wash 2 DeepWell source plate seated on silver insert SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 101 4 102 Hybridization 16 Load the BenchCel Microplate Handling Workstation according to Table 56 Table 56 Initial BenchCel configuration for SureSelectCapture amp Wash_v1 0 rst No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 2 Tip boxes Empty Empty Empty 3 3 Tip boxes Empty Empty Empty 4 4 Tip boxes Empty Empty Empty 6 6 Tip boxes Empty Empty Empty 12 11 Tip boxes Empty Empty Empty Run VWorks runset SureSelectCapture amp Wash_v1 0 rst 17 On the SureSelect setup form under Select Protocol to Run select SureSelectCapture amp Wash_v1 0 rst 18 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate containing the DNA samples at position 6 19 Select the number of columns of samples to be processed Runs must include 1 2
92. rrent state Bravo 1 Error There appears to be a plate present in or in frontof the 4 gripper s plate presence sensor kS Choose Retry to check the plate presence sensor Choose Ignore to continue to home theG axis Please note that any plate currently held by the Gripper will be dropped Choose Abort to cancel initialization SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 2 25 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation VWorks Automation Control Software 2 If you encounter the W axis error message shown below select Retry Please verify that itis safe to home a aspirate dispense axis If there is fluid in the tips you kS may want to manually home the W axis in diagnostics over a waste position Choose Retry to continue homing the W axis Choose Ignore to leave the W axis unhomed Choose Abort to cancel initialization Ignore and Continue leaving device in current state Abort 26 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Verifying the Simulation setting VWorks software may be run in simulation mode during which commands entered on screen are not completed by the NGS workstation If workstation devices do not respond when you start a run
93. s Runs Runs Tip boxes filled 2 3 4 5 7 14 Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 3 3 3 3 3 3 96 Eppendorf twin tec full skirt 4 4 4 4 4 4 plates Thermo Scientific Reservoirs 2 2 2 2 2 2 Axygen square well plate waste 1 1 1 1 1 1 Pre CapturePCR_RNASeq_ILM_v1 0 pro Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 1 1 1 1 Empty tip boxes for waste tips 1 1 1 1 1 1 PCR plates compatible with 1 1 1 1 1 1 thermal cycler AMPureXP_v1 1 pro Pre Capture PCR Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 2 2 3 6 Empty tip boxes for waste tips 1 1 1 1 1 1 Nunc DeepWell Plates 2 2 2 2 2 2 96 Eppendorf twin tec full skirt 1 1 1 1 1 1 plates Thermo Scientific Reservoirs 2 2 2 2 2 2 Axygen square well plate waste 1 1 1 1 1 1 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 147 6 Reference Aliquot_Libraries_v1 0 pro Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 1 1 1 1 Empty tip boxes for waste tips 1 1 1 1 1 1 PCR plates compatible with 1 1 1 1 1 1 thermal cycler SureSelectHybridization_v1 0 pro Labware 1 Column 2 Column 3 Column 4 Column 6 Column 12 Column Runs Runs Runs Runs Runs Runs Tip boxes filled 1 1 2 2 3 5
94. s Shelf 5 Shelf 4 Parameters Sars 1 Select Protocol to Run mRNA_Purification_v1 0 pro gt Shelf2 AMPureXP Case Shefi 2 Select PCR Plate labware for Thermal Cycling 96 ABI PCR half skirt in Red Alum Insert x imni 3 Select Number of Columns of Samples i z U 4 Click button below to Display Initial Workstation Setup Rog ES lt Position 3 gt Display Initial Clear Workstation Workstation Setup Setup Display lt Pos 4 Peltier gt __ lt Pos 5 Shaker gt lt Pos 6 Peltier gt Controls l Once you have loaded labware according to Workstation Setup on right click Run Selected Protocol to start run 5 Load labware according to Workstation Setup lt Pos 7 Maanetic gt lt Position8 gt W lt Pos 9 Chiller gt EE D ae lt Pos 7 Magnetic gt lt Position 8 gt Pos 9 Chiller gt Protocol Full Screen Gantt Chart Elapsed Time 00 00 00 Reset All Form Selections to Defaults Information Currently Running Protocol i BenchCel BenchCel Stacker 1 BenchCel Stacker 2 BenchCel Stacker 3 BenchCel Stacker 4 1 Open the form using the SureSelect_RNA_ILM VWForm shortcut on your desktop 2 Use the drop down menus on the form to select the appropriate SureSelect workflow step and number of columns of samples for the run 3 Once all run parameters have been specified on the form click Display Initial Workstation Setup Display Initial Workstation Setup
95. sh i User data entry Pause and Diagnose Continue 26 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 27 Quickly transfer the plate back to the thermal cycler held at 65 C After transferring the plate click Continue on the VWorks screen 28 To finish the VWorks protocol click Continue in the Unused Tips and Empty Tip box dialogs and click Yes in the Protocol Complete dialog CAUTION The temperature of the plate in the thermal cycler should be held at 65 C using a heated lid at 105 C The lid of the thermal cycler is hot and can cause burns Use caution when working near the lid 29 Incubate the hybridization mixture in the thermal cycler for 24 hours at 65 C with a heated lid at 105 C If you are using the SureCycler thermal cycler place a compression mat over the PCR plate before closing the thermal cycler lid for the 24 hour incubation period SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 97 4 Hybridization Step 3 Capture the hybridized DNA In this step the cDNA capture library hybrids are captured using streptavidin coated magnetic beads This step is run immediately after the 24 hour hybridization period This step is automated by the NGS workstation using the SureSelectCapture amp Wash_v1 0 rst runset with a total duration of approximately 3 hours A workstation operator must be present to
96. sted in Table 52 The volumes below include the required overage Table 52 Components required for magnetic bead washing procedure Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2 Columns 3Columns 4 Columns 6 Columns 12 Columns M 270 50 uL 425 uL 825 uL 1225 uL 1 65 mL 2 5 mL 5 0 mL Streptavidin bead suspension SureSelect 0 2 mL 1 7 mL 3 3 mL 4 9 mL 6 6 mL 10 mL 20 mL Binding Buffer Total Volume 0 25 mL 2 125 mL 4 125 mL 6 125 mL 8 25 mL 12 5 mL 25 mL b Mix the beads on a vortex mixer for 5 seconds c Put the vial into a magnetic device such as the Dynal magnetic separator d Remove and discard the supernatant e Repeat step a through step d for a total of 3 washes Retain the beads after each wash and combine with a fresh aliquot of the indicated volume of SureSelect Binding Buffer SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 99 4 Hybridization 6 Resuspend the beads in SureSelect Binding buffer according to Table 53 below Table 53 Preparation of magnetic beads for SureSelectCapture amp Wash_v1 0 rst Reagent Volume for Volumefor Volumefor Volumefor Volumefor Volumefor Volume for 1 Library 1 Column 2Columns 3Columns 4Columns 6 Columns 12 Columns SureSelect 0 2 mL 1 7 mL 3 3 mL 4 9 mL 6 6 mL 10 mL 20 mL Binding Buffer 100 7 Prepare a Nunc DeepWell source plate for the washed streptavidin bead suspension Fo
97. t of total RNA used for library prep Cycle Number 200 ng 2 ug 11 13 2 1 ug 4 ug 9 11 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 73 3 Sample Preparation Step 5 Purify amplified DNA using AMPure XP beads In this step the Agilent NGS Workstation transfers AMPure XP beads and amplified cDNA libraries to a Nunc DeepWell plate and then collects and washes the bead bound DNA Prepare the workstation and reagents 1 Clear the Labware MiniHub and BenchCel of all plates and tip boxes 2 Gently wipe down the Labware MiniHub Bravo deck and BenchCel with a NucleoClean decontamination wipe 3 Turn on the ThermoCube set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol 4 Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time 5 Mix the bead suspension well so that the reagent appears homogeneous and consistent in color 6 Prepare a Nunc DeepWell source plate for the beads by adding 65 uL of homogeneous AMPure XP beads per well for each well to be processed 7 Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water 8 Prepare a separate Thermo Scientific reservoir containing 45 mL of freshly prepared 70 ethanol 74 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 9 Load the
98. te types and ensure that the thermal cycler to be used is compatible with one of the supported PCR plate types Prepare and load the Agilent NGS Workstation as detailed in each of the protocol steps before initiating each automated protocol run When loading plates in the workstation s Labware MiniHub always place plates in the orientation shown in Figure 4 on page 38 To prevent contamination of reagents by nucleases always wear powder free laboratory gloves and use dedicated solutions and pipettors with nuclease free aerosol resistant tips Avoid repeated freeze thaw cycles of stock and diluted RNA and cDNA solutions Possible stopping points where samples may be stored at 20 C are marked in the protocol Do not subject the samples to multiple freeze thaw cycles When preparing master mix reagent stock solutions for use 1 Thaw the reagent vial as rapidly as possible without heating above room temperature 2 Mix on a vortex mixer then spin in a centrifuge for 5 to 10 seconds to drive the contents off of walls and lid 3 Store vials used during an experiment on ice or in a cold block 4 Library Preparation Master Mixes should not be frozen and thawed more than five times If you plan to use the reagents in more than five experiments aliquot to multiple vials to minimize freeze thaw cycles for each vial In general follow Biosafety Level 1 BL1 safety rules SureSelect Automated RNA Library Prep and Target Enrichment
99. ted RNA Library Prep and Target Enrichment for Illumina Sequencing Table 73 SureSelect Target Enrichment Box 2 Content Kit Component Reference 6 Details SureSelect Hyb 3 SureSelect Indexing Block 1 SureSelect Block 2 SureSelect ILM Indexing Block 3 SureSelect RNase Block tube with yellow cap tube with green cap tube with blue cap tube with brown cap tube with purple cap Table 74 Plate map for SSEL 8bp Indexes A01 through H12 provided in blue plate in Library Prep kit p n 5500 0135 1 2 3 4 5 6 7 8 9 10 11 12 A A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 B B01 B02 B03 B04 B05 B06 B07 B08 B09 B10 B11 B12 C C01 C02 C03 C04 C05 C06 C07 C08 c09 C10 C11 C12 D D01 D02 D03 D04 D05 D06 D07 D08 D09 D10 D11 D12 E E01 E02 E03 E04 E05 E06 E07 E08 E09 E10 E11 E12 F F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 G G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 H H01 H02 H03 H04 H05 H06 H07 H08 H09 H10 H11 H12 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 133 6 Reference Nucleotide Sequences of SureSelect Indexes A01 to H12 Each index is 8 nt in length See page 56 for sequencing run setup requirements for sequencing libraries using 8 bp indexes Table 75 SureSelect RNA Seq Indexes for indexing primers in blue 96 well plate Sequence AATGTTGC TGAAGAGA C10 AGATCGCA D10 AAGAGATC E10 CAACCACA F10 TGGAACAA G10 CCTCTATC H10 ACAGATTC A11 CCAGTTCA B11 TGGCTTCA
100. tes i I 4 C Hold When finished dick Continue below User data entry Pause and Diagnose Continue C a Remove the plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 seconds b Briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid c Transfer the PCR plate to a thermal cycler with the heated lid ON and run the RNA fragmentation program shown in Table 15 After transferring the plate click Continue on the VWorks screen Table 15 Thermal cycler program for RNA fragmentation Step Temperature Time Step 1 94 C 8 minutes Step 2 4 C Hold SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 30 During the 8 minute incubation step prepare the reagents and workstation for first strand cDNA synthesis a When prompted by the dialog below place a fresh PCR plate use the plate type specified during the run setup at position 6 seated in the red insert Proceed immediately to step b below Add Plate amp Add 1st Strand MM Place a new plate at position 6 and ensure that the same plate type is used Apply the appropriate amount of First Strand cDNA Master Mix with Actinomycin D to column 2 of the master mix plate at position 9 When finished click Continue below User data entry b Prepare a
101. the RNA fragmentation program Table 15 remove the plate from the thermal cycler and briefly spin in a centrifuge or mini plate spinner to collect the liquid b Place the RNA sample plate on position 4 of the Bravo deck seated in the red insert c Carefully unseal the plate then click Continue 52 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 32 The workstation removes the fragmented RNA samples from the bead containing wells and combines the samples with RNA Seq First Strand Master Mix Actinomycin D When the workstation has finished you will be prompted by VWorks as shown below 1st Strand cDNA Synthesis Get plate from position 6 seal at 165 C for 1 0s Place in thermal cycler using the following program 25 C for 10 minutes i 37 C for 40 minutes 4 C Hold When finished click Continue below User data entry Pause and Diagnose Continue C a Remove the plate from position 6 of the Bravo deck and seal the plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 seconds b Briefly spin the plate in a centrifuge or mini plate spinner to collect the liquid c Transfer the PCR plate to a thermal cycler with the heated lid ON and run the first strand cDNA synthesis program shown in Table 19 After transferring the plate click Continue on the VWorks screen Table 19 Thermal c
102. the plate to mix samples in each well then centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal Prepare the TapeStation samples as instructed in the Agilent 2200 TapeStation User Manual Use 2 uL of each indexed DNA sample diluted with 2 uL of High Sensitivity D1000 sample buffer for the analysis Make sure that you thoroughly mix the combined DNA and High Sensitivity D1000 sample buffer on a vortex mixer for 5 seconds for accurate quantitation Stopping Point 122 Load the sample plate or tube strips from step 3 the High Sensitivity D1000 ScreenTape and loading tips into the 2200 TapeStation as instructed in the Agilent 2200 TapeStation User Manual Start the run For each sample measure the concentration of the library ng uL by integrating under the peak at approximately 200 to 700 bp A sample electropherogram is shown in Figure 14 If you do not continue to the next step seal the plate and store at 20 C SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Indexing and Sample Prep for Multiplexed Sequencing Step 3 Assess DNA quality and quantity Lower o Sample Intensity FU 2 3 1500 100 g 8 g Figure 14 Analysis of purified indexed DNA amplicons using the 2200 TapeStation SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 5 bp 123 5 Indexing and S
103. tinue 21 Seal the sample plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 3 0 sec 22 Transfer the sealed plate to a thermal cycler and run the following program shown in Table 50 After transferring the plate click Continue on the VWorks screen Table 50 Thermal cycler program used for sample denaturation prior to hybridization Step Temperature Time Step 1 95 C 5 minutes Step 2 65 C Hold While the sample plate incubates on the thermal cycler the Agilent NGS Workstation combines aliquots of the SureSelect Capture Library master mix and Hybridization Buffer master mix 94 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Hybridization 4 CAUTION You must complete step 23 to step 27 quickly and immediately after being prompted by the VWorks software It is important that sample temperature remains approximately 65 C during transfers between the Agilent NGS Workstation and thermal cycler 23 When the workstation has finished aliquoting the Capture Library and Hybridization Buffer master mixes you will be prompted by VWorks as shown below When the thermal cycler reaches the 65 C hold step click Continue Leave the sample plate in the thermal cycler until you are notified to move it m Wait for plate in thermocycler When thermocyder has reached hold step at 65C dick Continue Leave DNA plate in thermocyder until you ar
104. tion for Kits with Original Index Configuration indexing primers in clear plate Use the reference information in this section if your kit includes Library Prep Kit p n 5500 0117 If your kit does not include this component kit see page 130 for kit content and indexing primer information Kit Contents The SureSelect RNA Reagent Kits contain the following component kits Table 76 SureSelect RNA Seq Kit Content Original Index Configuration Component Kits Storage Condition Part Number SureSelect Strand Specific RNA Library 20 C 5500 0117 Prep ILM Box 1 SureSelect Strand Specific RNA Library 4 C 5190 6411 Prep ILM Box 2 SureSelect Target Enrichment Box 1 Room Temperature 5190 4394 SureSelect Target Enrichment Box 2 20 C 5190 6262 SureSelect capture libraries and reagents must be used within one year of receipt SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 135 6 Reference The contents of each of the component kits listed in Table 76 are described in the tables below Table 77 SureSelect Strand Specific RNA Library Prep ILM Box 1 Content Original Index Configuration Kit Component Details RNA Seq Fragmentation Mix bottle RNA Seq First Strand Master Mix tube with orange cap RNA Seq Second Strand End Repair Enzyme Mix bottle RNA Seq Second Strand End Repair Oligo Mix tube with yellow cap RNA Seq dA Tailing Master Mix bottle SureSelect Ligation Master Mix
105. tion of the master mix source plate for SureSelectHybridization_v1 0 pro 10 Seal the master mix source plate using the PlateLoc Thermal Microplate Sealer with sealing settings of 165 C and 1 0 sec 11 Centrifuge the plate for 30 seconds to drive the well contents off the walls and plate seal and to eliminate any bubbles Keep the master mix plate at room temperature SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 91 4 92 Hybridization Load the Agilent NGS Workstation 12 Load the Bravo deck according to Table 48 Table 48 Initial Bravo deck configuration for SureSelectHybridization_v1 0 pro Location Content 5 Empty Eppendorf twin tec plate 6 100 ng aliquots of prepped DNA libraries in PCR plate seated on red insert PCR plate type must be specified on setup form under step 2 8 Empty tip box 9 Hybridization Master Mix source plate seated on silver insert 13 Load the BenchCel Microplate Handling Workstation according to Table 49 Table 49 Initial BenchCel configuration for SureSelectHybridization_v1 0 pro No of Columns Rack 1 Rack 2 Rack 3 Rack 4 Processed 1 1 Tip box Empty Empty Empty 2 1 Tip box Empty Empty Empty 3 2 Tip boxes Empty Empty Empty 4 2 Tip boxes Empty Empty Empty 6 3 Tip boxes Empty Empty Empty 12 5 Tip boxes Empty Empty Empty Run VWorks protocol SureSelectHybridization_v1 0 pro 14 On the SureSelect setup form under Select Protocol to Run sele
106. tion to an operating procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 3 In this Guide This guide describes an optimized protocol to prepare target enriched mRNA sequencing libraries from total RNA samples using the SureSelect Automated Strand Specific RNA Library Prep and Target Enrichment system This protocol is specifically developed for RNA library preparation for Illumina paired end multiplexed sequencing Sample processing steps are automated using the NGS Workstation Before You Begin This chapter contains information such as procedural notes safety information required reagents and equipment that you should read and understand before you start an experiment Using the Agilent NGS Workstation for SureSelect RNA Library Preparation This chapter contains an orientation to the Agilent NGS Workstation an overview of the SureSelect RNA protocol and c
107. to incubate components at a defined temperature during the run During protocols that require temperature control at position 9 you will be instructed to start and set the temperature of the ThermoCube device before starting the run ThermoCube temperature settings are modified using the control panel LCD display screen and four input buttons on the front panel of the device using the following steps 1 Turn on the ThermoCube and wait for the LCD screen to display TEMP 2 Press the UP or DOWN button to change SET TEMP 1 to the required set point 3 Press the START button The ThermoCube will then initates temperature control of Bravo deck position 9 at the displayed set point SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 21 2 Using the Agilent NGS Workstation for SureSelect RNA Library Preparation VWorks Automation Control Software VWorks software included with your Agilent NGS Workstation allows you to control the robot and integrated devices using a PC The Agilent NGS Workstation is preloaded with VWorks software containing all of the necessary SureSelect system liquid handling protocols General instructions for starting up the VWorks software and the included protocols is provided below Each time a specific VWorks protocol is used in the SureSelect procedure any settings required for that protocol are included in the relevant section of this manual The instructions in this man
108. to wells of the source plate corresponding to the total RNA sample wells in step 7 above For example for 3 column runs fill source well plate wells Al to H3 but leave wells A4 to H12 empty 8 Prepare the oligo dT beads source plate a Vortex the Oligo dT Microparticles until the suspension appears homogeneous and consistent in color If bead aggregates are still present after vortexing mix thoroughly by pipetting up and down until the suspension appears homogeneous b Ina PCR plate that is compatible with the thermal cycler to be used in the run place 25 uL of the homogeneous Oligo dT bead suspension into each well to be used for sample purification 9 Prepare the Bead Binding Buffer source plate Place 30 uL of RNA Seq Bead Binding Buffer into wells of a 96 well Eppendorf twin tec plate Fill each well that corresponds to an RNA sample well 10 Prepare the Bead Elution Buffer source plate Place 30 uL of RNA Seq Bead Elution Buffer into wells of a 96 well Eppendorf twin tec plate Fill each well that corresponds to an RNA sample well 11 Prepare the Bead Wash Buffer source plate Place 410 uL of RNA Seq Bead Washing Buffer into wells of a Nunc DeepWell plate Fill each well that corresponds to an RNA sample well 36 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Sample Preparation 3 Prepare the master mix source plate 12 Prepare the master mix source plate by adding the appropriate volu
109. tube with purple cap SureSelect Oligo Adaptor Mix tube with blue cap RNA Seq PCR Master Mix bottle Uracil DNA Glycosylase UDG tube with yellow cap SureSelect Primer tube with brown cap RNA Seq ILM Reverse PCR Primer tube with black cap RNA Seq ILM Post capture PCR Primer tube with green cap RNA Seq Indexes 8 bp RNA Seq Indexes 1 96 8 bp provided in clear 96 well plate See Table 82 on page 140 through Table 87 on page 145 for index sequence information Tt See Table 81 on page 139 for a plate map 136 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Reference 6 Table 78 SureSelect Strand Specific RNA Library Prep ILM Box 2 Content Kit Component Details Oligo dT Microparticles bottle RNA Seq Bead Binding Buffer bottle RNA Seq Bead Washing Buffer bottle RNA Seq Bead Elution Buffer bottle Nuclease Free Water bottle Table 79 SureSelect Target Enrichment Box 1 Content Kit Component Details SureSelect Hyb 1 tube with orange cap SureSelect Hyb 2 tube with red cap SureSelect Hyb 4 tube with black cap SureSelect Binding Buffer bottle SureSelect Wash Buffer 1 bottle SureSelect Wash Buffer 2 bottle SureSelect Elution Buffer bottle SureSelect Neutralization Buffer bottle The provided SureSelect Elution Buffer and Neutralization Buffer are not used in the auto mated RNA Target Enrichment workflow described in this manual SureSelect Automated RN
110. ual are compatible with VWorks software version 11 3 0 1195 If you have questions about VWorks version compatibility please contact service automation agilent com Logging in to the VWorks software 1 Double click the VWorks icon or the SureSelect_RNA_ILM VWForm shortcut on the Windows desktop to start the VWorks software 2 If User Authentication dialog is not visible click Log in on the VWorks window toolbar 3 In the User Authentication dialog type your VWorks user name and password and click OK If no user account is set up contact the administrator VWorks protocol and runset files VWorks software uses two file types for automation runs pro protocol files and rst runset files Runset files are used for automated procedures in which the workstation uses more than one automation protocol during the run 22 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing Using the Agilent NGS Workstation for SureSelect RNA Library Preparation 2 Using the SureSelect_RNA_ILM VWForm to setup and start a run Use the VWorks form SureSelect_RNA_ILM VWForm shown below to set up and start each SureSelect automation protocol or runset E Works SureSelect RNAILM WWForm E a nO Om gt Workstation Setup lt Position 1 gt lt Position 2 gt MiniHub SureSelect RNA MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 MiniHub Cassette 4 for Illumina sequencer
111. used on the SureSelect_RNA_ILM VWForm to allow correct configuration of the liquid handling components for the PCR plate type Before you begin the automation protocol make sure that you are using a supported PCR plate type The PCR plate type to be used in the protocol is specified using the menu below Vendor and part number information is provided for the supported plate types in Table 7 2 Select PCR Plate labware for Thermal Cycling 96 ABI PCR half skirt in Red Alum Insert 3 Eas half skirt in Red Alum Insert 96 Agilent Semi skirted PCR in Red Alum Insert 96 Eppendorf Twin tec half skirt PCR in Red Alum Insert 4 96 Eppendorf Twin tec PCR in Red Alum Insert Table 7 Ordering information for supported PCR plates Description in VWorks menu Vendor and part number 96 ABI PCR half skirted plates MicroAmp Optical Life Technologies p n N8010560 plates 96 Agilent semi skirted PCR plate Agilent p n 401334 96 Eppendorf Twin tec half skirted PCR plates Eppendorf p n 951020303 96 Eppendorf Twin tec PCR plates full skirted Eppendorf p n 951020401 or 951020619 32 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing SureSelect Automated Strand Specific RNA Target Enrichment Protocol c D ee 3 7 e Sample Preparation Step 1 Purify poly A RNA fragment RNA synthesize first strand cDNA 34 Step 2 Purify first strand cDNA using AMPure XP beads 54 Step 3 Pre
112. x Empty Empty Empty 12 1 Tip box Empty Empty Empty Run VWorks protocol Post CapturePCR_RNASeq_ILM_v1 0 pro 13 On the SureSelect setup form under Select Protocol to Run select Post CapturePCR_RNASeq_ILM_v1 0 pro 14 Under Select PCR plate labware for Thermal Cycling select the specific type of PCR plate containing the indexing primers at position 6 15 Select the number of columns of samples to be processed Runs must include 1 2 3 4 6 or 12 columns 16 Click Display Initial Workstation Setup Display Initial Workstation Setup 17 Verify that the NGS workstation has been set up as displayed in the Workstation Setup region of the form Workstation Setup a MiniHub t MiniHub Cassette 1 MiniHub Cassette 2 MiniHub Cassette 3 Mini Shelf 5 4 A ShA Aana we lt a gae oe ee ee SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 113 5 Indexing and Sample Prep for Multiplexed Sequencing 18 When verification is complete click Run Selected Protocol p Run Selected Protocol Running the Post CapturePCR_RNASeq_ILM_v1 0 pro protocol takes approximately 15 minutes Once complete the PCR ready samples containing captured DNA indexing primer and PCR master mix are located in the PCR plate at position 6 of the Bravo deck The Eppendorf plate containing the remaining bead bound captured DNA samples which may be stored for future use at 4 C overnight or at 20 C for longer
113. y and quality 80 SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing SureSelect Automated Strand Specific RNA Target Enrichment Protocol ee 4 CAUTION 070 Hybridization ss Step 1 Aliquot prepped DNA libraries for hybridization 82 Step 2 Hybridize the DNA library and SureSelect RNA Capture Library 85 Step 3 Capture the hybridized DNA 98 This chapter describes the steps to combine the prepped library with the blocking agents and the SureSelect RNA capture library Each cDNA library sample must be hybridized and captured individually prior to addition of the indexing tag by PCR The ratio of SureSelect capture library to prepped library is critical for successful capture CAUTION You must avoid evaporation from the small volumes of the capture during the 24 hour incubation If you want to use a duration of hybridization gt 24 hours first test the conditions Incubate 35 uL of SureSelect Hybridization Buffer without DNA at 65 C for 24 hours or longer if applicable as a test Include buffer in each well that you might use including those in the center and those on the edges Check that you do not get extensive evaporation Evaporation should not exceed 3 to 4 pL ait Agilent Technologies 81 4 Hybridization Step 1 Aliquot prepped DNA libraries for hybridization For each sample library prepared do one hybridization and capture Do not pool samples
114. ycler program for first strand cDNA synthesis Step Temperature Time Step 1 25 C 10 minutes Step 2 37 C 40 minutes Step 3 4 C Hold SureSelect Automated RNA Library Prep and Target Enrichment for Illumina Sequencing 53 3 Sample Preparation Step 2 Purify first strand cDNA using AMPure XP beads In this step the Agilent NGS Workstation transfers AMPure XP beads and first strand cDNA samples to a Nunc DeepWell plate and then collects and washes the bead bound DNA Prepare the workstation and reagents 1 Leave tip boxes on shelves 1 and 2 in casette 1 of the Labware MiniHub from the previous mRNA_Purification_v1 0 pro run Otherwise clear the remaining positions of the MiniHub and BenchCel of plates and tip boxes Gently wipe down the Labware MiniHub Bravo deck and BenchCel with a NucleoClean decontamination wipe Turn on the ThermoCube set to 0 C at position 9 of the Bravo deck Be sure that the chiller reservoir contains at least 300 mL of 25 ethanol Let the AMPure XP beads come to room temperature for at least 30 minutes Do not freeze the beads at any time Mix the bead suspension well so that the reagent appears homogeneous and consistent in color Prepare a Nunc DeepWell source plate for the beads by adding 51 uL of homogeneous AMPure XP beads per well for each well to be processed Prepare a Thermo Scientific reservoir containing 15 mL of nuclease free water Prepare a separate Thermo Scientifi

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