Encore® SP+ Complete Library Systems
Contents
1. 15 briefly Place on ice Use immediately Encore SP Complete Library Systems using them as they will be used again in the Agencourt RNAClean XP Bead Master Mix Each Sample Solution Mix should be mixed well and incubated at room temperature approximately 23 C for 10 minutes The above recipe is meant for a single sample Prepare one Sample Solution Mix for each sample to be loaded into the cartridge 2 Encore SP Complete Reagent Master Mix Preparation Prepare Bead Binding Solution Bead Wash Solution and Elution Buffer 1 Remove the Bead Binding Solution Bead Wash Solution and Elution Buffer from room temperature reagent box 2 Vortex to mix and spin down briefly Leave tubes at room temperature while pre paring the additional master mixes below Prepare DR Multiplex Barcode Adaptors 1 Thaw DR Multiplex Ligation Adaptor Mixes L2V10DR BC1 8 or L2V10DR BC9 16 at room temperature 2 Vortex well to mix and spin down briefly Leave tubes at room temperature while preparing the additional master mixes below Prepare First Strand Primer Mix and First Strand cDNA Master Mix 1 Remove the First Strand Primer Mix blue A1 ver 11 from 20 C storage spin down briefly and place on ice to thaw Leave the First Strand Primer Mix on ice until ready to load into the Mondrian SP Cartridge See Step 2 of the Mondrian SP Cartridge Loading Instructions below 2 Thaw the First Strand Buffer Mix blue A2 ver 9 at room temper2. 2 Add RNA sample up to 500 ng and add Nuclease free Water D1 green cap to bring the final volume to 25 pL 3 Incubate at 25 C for 15 minutes followed by 37 C for 15 minutes and return to ice 4 After the DNase treatment the sample must be purified We recommend either of the two purification procedures below Purification with RNA Clean amp Concentrator 5 Zymo Research Cat R1015 e Add 4 volumes 100 uL of RNA binding buffer to the sample e Obtain one RNA Clean amp Concentrator 5 Kit column and apply the sample to the column e Spin column for 30 seconds at 28000 X g 210 000 rpm Discard the flow through e Add 200 uL wash buffer with ethanol added as per vendor s specifications e After closing the column spin for 30 seconds at gt 8000 X g 210 000 rpm Discard the flow through e Add 200 uL fresh 80 ethanol close cap and spin for 30 seconds at 28000 X g 210 000 rpm Discard the flow through e Place the RNA Clean amp Concentrator 5 Kit column in a fresh 1 5 mL collection tube e Add 10 pL Nuclease free Water green D1 directly to the center of the filter in the tube and close the cap Important Allow the nuclease free water to come to room temperature prior to use e Spin for 1 minute at 28000 X g 210 000 rpm to collect the purified RNA VII Appendix 28 Encore SP Complete Library Systems Purification with QIAGEN RNeasy MinElute Cleanup Columns QIAGEN Cat 74204
3. Add 80 uL ice cold Nuclease free Water D1 green cap to the sample on ice Add 350 uL Buffer RLT and mix by pipetting Add 250 uL 96 to 100 ethanol and mix thoroughly by pipetting Place an RNeasy MinElute Spin Column into a 2 mL collection tube one column per sample and apply the 700 uL sample to the column After closing the column spin for 15 seconds at 28000 X g 210 000 rpm Discard the flow through Place the RNeasy MinElute Spin Column into a fresh 2 mL collection tube Add 500 pL Buffer RPE to the column and close the tube Spin for 15 seconds at gt 8000 X g 210 000 rpm Discard the flow through keeping the same collec tion tube Add 500 uL 80 ethanol to the RNeasy MinElute Spin Column and close the tube Note Use fresh 80 ethanol Lower percent ethanol mixes will reduce recovery Spin for 2 minutes at gt 8000 X g 210 000 rpm Discard the flow through Place the RNeasy MinElute Spin Column in a fresh 2 mL collection tube and place in the microcentrifuge with the cap open Spin for 5 minutes at 28000 X g 210 000 rpm and discard the flow through Place the RNeasy MinElute Spin Column in a fresh 1 5 mL collection tube Add 14 uL Nuclease free Water D1 green cap directly to the center of the filter in the tube and close the cap Do not use cold water Spin for 1 minute at 28000 X g 210 000 rpm to collect the purified RNA B Mondrian SP Cartridge Handling Background The Mondrian SP Cartridge is use
4. Ensure that the reagent is carefully dispensed at the very bottom of the port Adaptor ports A1 through A8 are highlighted with yellow rims Scocooo Adaptors 2 Load 25 uL Strand Selection II Library Amplification Master Mix tube E1 into E1 Port E1 is highlighted with a light blue rim Oz 3 Vortex the Agencourt RNAClean XP Bead Master Mix tube E2 to ensure the beads are resuspended and load 30 uL Bead Master Mix into E2 Port E2 is highlighted with a purple rim Or 20 Encore SP Complete Library Systems IV Protocol 21 Encore SP Complete Library Systems 4 Load 50 uL Bead Binding Solution into E3 and E4 Ports E3 and E3 E4 E4 are not highlighted in any way O O 5 If Cartridge QC has been completed skip this step and proceed to E5 step 6 If Cartridge QC has not been completed load 50 pL Elution Buffer into E5 Port E5 is highlighted with a grey rim 6 Load 50 pL Bead Wash Solution into E6 and E7 Ports E6 and E7 are E E7 highlighted with a black rim OO 7 Load 8 uL First Strand Primer Mix blue A1 ver 11 into D1 Port D1 is D1 highlighted with a brown rim O 8 Load 8 uL First Strand cDNA Master Mix tube D2 into D2 Port D2 is D2 highlighted with a yellow rim O 9 Load 8 uL Second Strand cDNA Master Mix tube D3 into D3 Port D3 D3 is highlighted with a red rim O 10 Load 8 uL Strand Selection Master Mix tube D4 into D4 Port D4 is D4 hig
5. any Duplex Set 3 of the other remaining L2V10DR BC GIGCTIT six individual barcodes L2V10DR BC7 AAGCCT Duplex Set 4 L2V10DR BC8 GTCGTA VII Appendix 34 Encore SP Complete Library Systems Table 13 Barcode sequences for dedicated read DR adaptors used in Encore SP Complete Multiplex System 9 16 Part No 8152 Barcode eee 6 nt Barcode Adaptor Pairing Barcoce Adaptor Ligation Pairing for 3 plex 7 Sequence as Read for 2 plex Adaptor Mix Through 8 plex by the Sequencer Multiplex i Sequencing Multiplex Sequencing L2V10DR BC9 AAGAGG Duplex Set 1 L2V10DR BC10 GGAGAA L2V10DR BC11 AGCATG One of the duplex sets VIODRIBCI2 GAGTCA Duplex Set 2 from the column to the left must be used in 2V10DR BC13 CGTAGA combination with any E Duplex Set 3 of the other remaining L2V10DR BC14 TCAGAG six individual barcodes L2V10DR BC15 CACAGT Duplex Set 4 L2V10DR BC16 TTGGCA For additional information on the design strategy and sequence of the barcode adap tors please refer to the FAQs in Appendix D VII Appendix 35 Encore SP Complete Library Systems E Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 What materials are provided with the Encore SP Complete Library Systems The Encore SP Complete Library Systems kits provide all necessary buffers primers enzymes and purification beads Does this syste
6. micro centrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 10 Label the tube E1 Table 10 Strand Selection II Library Amplification Master Mix COMPONENT VIAL CAP VOLUME Strand Selection II Amplification Buffer Mix SS3 P1 Red 17 5 ul Strand Selection Enzyme II Mix SS4 Purple 2 5 uL Amplification Primer Mix P2 ver 9 Red 2 0 uL Amplification Enzyme Mix P3 ver 2 Red 6 0 uL Total volume 28 0 pL Prepare Bead Master Mix 1 Vortex the Agencourt RNAClean XP Beads which have been resting at room tem perature and vortex to mix well Keep at room temperature 2 Remove the Bead Additive from the room temperature box and vortex to mix well Keep at room temperature 3 Prepare the Bead Master Mix in a 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 11 Mix well by pipetting Label the tube E2 IV Protocol E Table 11 Bead Master Mix EY Mix well by pipetting COMPONENT VIAL CAP VOLUME Agencourt RNAClean XP Beads N A 35 0 uL Bead Additive N A 1 0 uL Total volume 36 0 pL 3 Mondrian SP Cartridge Loading Instructions For detailed instructions on how to prepare the Mondrian SP Cartridge for reagent and sample loading refer to Mondrian SP Cartridge User Guide M01344 Figure 3 Mondrian SP Cartridge loading guide for the Encore SP Complete Library System protocol Filler E1 E2 E3 E4
7. somewhat degraded samples With such samples users may experience lower yields and may encounter affected sequencing metrics Degraded samples must be empiri cally tested to determine their performance with the Encore SP Complete Library Systems Do I need to perform an rRNA depletion or Poly A enrichment step before processing RNA samples with the Encore SP Complete Library Systems No The system is designed to use total RNA as input rRNA depletion or Poly A enrichment is not necessary How much total RNA do need for Encore SP Complete 100 150 ng total RNA input VII Appendix 36 Encore SP Complete Library Systems Q9 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 How does your protocol improve the efficiency of ligation and avoid adaptor dimer formation The Encore SP Complete Library Systems use optimized chemistries to increase the efficiency of blunt end adaptor ligation and minimize the amount of adaptor dimer in the library How does your protocol enable strand retention The Encore SP Complete Library Systems use targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality What percentage of rRNA reads can expect in my data Use of high quality higher vertebrate total RNA including human rat and mouse has routinely achieved lt 25 of total reads aligning to rRNA and mitochondrial sequences In silico analysis suggests sim
8. 1 OOOOOOO D2 D3 D4 DS D D7 OOOQOQOQOOQO 99060600 OO0OO0OO0OO0OO0OO Adaptors Encore SP Complete DR Multiplex Systems Single use only gait NUGEN imagine more from less Sample Input 00086009 P01212 v1 19 Encore SP Complete Library Systems IV Protocol Loading Reagents and Samples in the Mondrian SP Cartridge Using Figure 3 as a guide follow the instructions below to load reagents into their appropriate cartridge ports e Use a 10 uL pipette to add DR Multiplex Ligation Adaptor Mixes First and Second Strand Master Mixes Ligation Master Mix End Repair Master Mix and Fragmentation Master Mix e Use a 100 or 200 uL pipette for adding the Sample Solution Mixes Strand Selection II Library Amplification Master Mix Bead Binding Solution Elution Buffer Bead Master Mix and Bead Wash Solution e Load the ports in a steady manner to avoid overflow of the Filler Fluid e When adding samples and reagents insert the pipette tip into the port until it contacts the bottom of the cartridge then withdraw the pipette tip slightly to allow space for dispensing Slowly depress the plunger to dispense the reagent but do not depress the plunger completely blow out as this could introduce bubbles into the cartridge 1 Load 1 5 pL of each DR Multiplex Ligation Adaptor Mix L2V10DR BC1 8 or L2V10DR BC9 16 into the appropriate port A1 through A8 matching the sample to be barcoded
9. 727 L2V10DR BC6 01728 L2V10DR BC7 01729 L2V10DR BC8 S01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 01718 Strand Selection Buffer Mix 1 of 2 Purple SS1 VER 2 01512 Strand Selection Enzyme Mix 1 of 2 Purple SS2 01738 Strand Selection Enzyme Mix II 1 of 2 Purple SS4 Strand Selection II 301907 Amplification Buffer Mix pele Reg aaa 01765 Amplification Primer Mix 1 of 2 Red P2 ver 9 6 Components Encore SP Complete Library Systems Encore SP Complete DR Multiplex System 1 8 Components and Reagents Part No 8151 continued ee 8151 8151 8151 soe NUMBER DESCRIPTION BOX VIAL CAP NUMBER S01764 Amplification Enzyme Mix 1 of 2 Red P3 ver 2 01001 Nuclease Free Water 1 of 2 Green D1 PO1208 Mondrian SP Cartridges x4 2 of 2 N A N A Encore SP Complete Library PO1212 Systems Cartridge Loading 2 of 2 N A N A Guide x4 01719 Mondrian SP Filler Fluid x4 2 of 2 N A N A S01587 sample Concentration 2of2 Clear N A Solution S01588 Bead Binding Solution 2of2 Clear N A 01589 Bead Wash Solution 2 of 2 Clear N A S 01590 Elution Buffer 2 of 2 Clear N A S01825 Bead Additive ie Clear N A separately 501698 Agencourt RNAClean XP Shipped Clear N A Beads separately Table 2 Encore SP Complete DR Multiplex System 9 16 Components and Re agents Part No 8152 8152 PART RON eae ore AMUSE NUMBER 01711 First Strand Primer Mix 1 of 2 Blue A1 v
10. 914 Canned air ce bucket Cleaning solutions such as RNaseZAP Life Technologies Cat AM9780 and DNA OFF MP Biomedicals Cat QD0500 OPTIONAL Products for DNase treatment and RNA template clean up QIAGEN MinElute Reaction Cleanup Kit QIAGEN Cat 28204 QIAGEN RNase Free DNase Set QIAGEN Cat 79254 QIAGEN RNeasy Mini RNA Purification Kit QIAGEN Cat 74104 RNA Clean amp Concentrator 5 Zymo Research Cat R1015 QIAGEN RNeasy MinElute Cleanup Columns QIAGEN Cat 74204 To Order e Affymetrix www affymetrix com e Agilent www agilent com e Lif e Technologies www lifetechnologies com e MP Biomedicals www mpbio com e Ol AGEN www qiagen com e VWR www vwr com e Zy mo Research www zymoresearch com Ill Planning the Experiment A Input RNA Requirements 1 RNA Quantity Total RNA input must be between 100 ng and 150 ng Inputs outside of this range may affect reaction stoichiometry resulting in sub optimal libraries Low input amounts may result in insufficient yields depending on the requirements of your analytical platform We strongly recommend quantitation of total RNA to ensure the minimum input requirement is met 2 RNA Purity RNA samples must be free of contaminating proteins and other cellular mate rial organic solvents including phenol and ethanol and salts used in many RNA isolation methods When preparing small amounts of RNA we recommend using a com
11. Encore SP Complete Library Systems PART NOS 8151 32 AND 8152 32 ga NUGEN imagine more from lesse Patents Licensing and Trademarks 2013 2014 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugen com NuUGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NUGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners Specific information on patents trademarks and licenses related to the Mondrian SP Universal Cartridge the Mondrian SP Cartridge the Mondrian SP Workstation and the Mondrian SP Workstation may be found in the Mondrian SP Universal Cartridge User Guide M01265 the Mondrian SP Cartridge User Guide M01344 the Mondrian SP Workstation User Manual Part No M01264 and the Mondrian SP Workstation User Manual M01322 The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to modify reverse engineer resell repackage or further sublicense under these patent applications and any patents issuing from these patent applications to use this prod uct and methods accompanying this user guide for research and development purposes solely in accor
12. GEN distributor for technical support VII Appendix 26 Encore SP Complete Library Systems A DNase Treatment of RNA DNase Treatment During Purification Using the QIAGEN RNase Free DNase Set and the RNeasy Mini RNA Purification Kit 1 Homogenize sample in RLT buffer including B mercaptoethanol according to the type of sample as described in the RNeasy Mini Kit protocol 2 Add 1X volume of 70 ethanol to the homogenized lysate pipet up and down to mix sample well Do not centrifuge 3 Place an RNeasy mini column in a 2 mL collection tube 4 Apply the sample up to 700 uL including any precipitate that may have formed to the column 5 Close the tube gently and centrifuge for 15 seconds at 28000 X g 210 000 rpm Discard the flow through 6 For volumes greater than 700 pL load aliquots onto the RNeasy column succes sively and centrifuge as before 7 Add 350 uL Buffer RW1 into the RNeasy mini column to wash and centrifuge for 15 seconds at gt 8000 X g 210 000 rpm Discard the flow through 8 Add 10 uL DNase to 70 uL Buffer RDD Gently invert the tube to mix Note Other DNase enzymes we recommend for use in this step are the Shrimp DNase recombinant from USB Corp use 10 uL or the DNase RNase free from New England BioLabs use 10 pL 9 Pipet the DNase incubation mix 80 pL directly onto the membrane inside the RNeasy mini column Incubate at the bench top 25 C for 15 minutes 10 A
13. ables 12 and 13 have an edit distance of three meaning that three events such as nucleotide substitu tion insertion or mutation must occur before any of the barcodes is con verted into another barcode sequence For example three different events edits would have to occur in the sequence of barcode L2V10DR BC4 before its sequence would be the same as any other barcode included in the Encore SP Complete Library Systems Users must decide whether or not to configure their barcode parsing software to accept barcode sequences with mismatches between the reference sequence of the barcode from Tables 12 and 13 and the actual sequence results For a detailed explanation of the error correction approach used for the DR barcodes please refer to Faircloth BC Glenn TC 2012 Not All Sequence Tags Are Created Equal Designing and Validating Sequence Identification Tags Robust to Indels PLoS ONE 7 8 e42543 doi 10 1371 journal pone 0042543 What kind of sequencing primers can use with libraries generated using the Encore SP Complete Library Systems The Encore SP Complete Library Systems are designed for use with the stan dard Illumina sequencing primers for both single end and paired end sequenc ing applications Can the Encore SP Complete Library Systems be used with paired end sequencing Yes They can be used for both single end and paired end sequencing Special consideration should be given to the expected insert size in the pa
14. and stored at 4 C e All other Box 2 components should be stored at room temperature The kit has been tested to perform to specifications after as many as four freeze thaw cycles Kits handled and stored according to the above guidelines will perform to specifi cations for at least six months E Material Safety Data Sheet MSDS An MSDS for this product is available on the NuGEN website at www nugen com nugen index cfm support user guides 4 Encore SP Complete Library Systems 5 Components Encore SP Complete Library Systems A Reagents Provided Table 1 Encore SP Complete DR Multiplex System 1 8 Part No 8151 32 zel 8151 8151 8151 Pi NUMBER DESCRIPTION BOX VIAL CAP NUMBER S01711 First Strand Primer Mix 1of2 Blue Al ver 11 01712 First Strand Buffer Mix 1 of 2 Blue A2 ver 9 01713 First Strand Enzyme Mix 1 of 2 Blue A3 ver 5 S01714 Second Strand Buffer Mix 1 of 2 Yellow B1 ver 7 01715 Second Strand Enzyme Mix 1 of 2 Yellow B2 ver 4 501717 Fragmentation Enzyme Mix 1 of 2 Orange F2 ver 3 S01716 Fragmentation Buffer Mix 1 of 2 Orange F1 ver 3 01627 End Repair Buffer Mix 1 of 2 Blue ER1 ver 5 01510 End Repair Enzyme Mix 1 of 2 Blue ER2 ver 4 01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 01722 DR Multiplex Ligation Adaptor 1 of 2 Yellow L2V10DR BC1 01723 Mixes 1 8 L2V10DR BC2 01724 L2V10DR BC3 01725 L2V10DR BC4 01726 L2V10DR BC5 01
15. ature and vortex to mix well Briefly spin down the First Strand Enzyme Mix A3 ver 5 and place on ice 3 Prepare the First Strand cDNA Master Mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 4 Label the tube D2 Table 4 First Strand cDNA Master Mix COMPONENT VIAL CAP VOLUME First Strand Buffer Mix A2 ver 9 Blue 7 7 uL First Strand Enzyme Mix A3 ver 5 Blue 2 3 uL Total volume 10 0 pL IV Protocol a Prepare Second Strand cDNA Master Mix 1 Remove the Second Strand Enzyme Mix yellow B2 ver 4 from 20 C storage spin down briefly and place on ice to thaw 2 Thaw the Second Strand Buffer Mix yellow B1 ver 7 at room temperature vortex to mix well and spin down briefly Place on ice 3 Prepare the Second Strand cDNA Master Mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 5 Label the tube D3 Table 5 Second Strand cDNA Master Mix Mix by pipetting and spin COMPONENT VIAL CAP VOLUME down the master mix briefly Place a See Second Strand Buffer Mix B1 ver 7 Yellow 6 0 uL immediately Second Strand Enzyme Mix B2 ver 4 Yellow 4 0 uL Total volume 10 0 pL Prepare Strand Selection Master Mix 1 Thaw the Strand Selection Buffer Mix purple SS1 ver 2 at room temp and vortex to mix well Keep Strand Selection Enzyme Mix purple SS2 on ice 2 Prepare the Strand Sele
16. ction Master Mix in a 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 6 Label the tube D4 Table 6 Strand Selection Master Mix Mix by pipetting and spin COMPONENT VIAL CAP VOLUME down the master mix briefly Place on ice Use Strand Selection Buffer Mix SS1 ver 2 Purple 8 5 uL immediately Strand Selection Enzyme Mix SS2 Purple 1 5 uL Total volume 10 0 pL Prepare Ligation Master Mix 1 Thaw Ligation Buffer Mix yellow L1 ver 5 at room temp and vortex to mix well Spin and place the Ligation Enzyme Mix yellow L3 ver 4 on ice 2 Prepare the Ligation Master Mix in a 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 7 Label the tube D5 16 Encore SP Complete Library Systems IV Protocol E Table 7 Ligation Master Mix Mix by pipetting and spin COMPONENT VIAL CAP VOLUME down the master mix aa ene Uae Ligation Buffer Mix L1 ver 5 Yellow 7 0 uL Ligation Enzyme Mix L3 ver 4 Yellow 3 0 uL Total volume 10 0 pL Prepare End Repair Master Mix 1 Thaw the End Repair Buffer Mix blue ER1 ver 5 at room temp and vortex to mix well Spin and place the End Repair Enzyme Mix blue ER2 ver 4 on ice 2 Prepare the End Repair Master Mix in a 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 8 Label the tube D6 Table 8 End Repair Master M
17. d with the Mondrian SP System and offers a convenient 8 sample batch size simple reagent loading and easy sample recovery Reagents are contained in discrete droplets encased within Filler Fluid isolating the reactions from the lab environment and carryover contamination Construction When prepared for use cartridges consist of an oil layer Filler Fluid sandwiched between a PCB substrate and a clear top plate The PCB substrate is patterned with insulated electrodes By changing the relative voltages of the patterned electrodes VII Appendix 29 Encore SP Complete Library Systems aqueous droplets are manipulated to perform complex assays Droplets are dispensed from loading ports transported to various locations on the cartridge mixed incubated and collected using only software control Figure 5 shows a cutaway side view sche matic of a cartridge Figure 5 Cartridge cutaway side view Top plate D lt f Droplet Filler fluid oil e a PCB substrate Insulating polymer Electrode Cartridges Cartridges are one time use only Cartridge re use is not supported even if a run is canceled prior to completion Figure 6 Top view of a Mondrian SP Cartridge Sample ports along the bottom edge are labeled S1 8 Other reagent port rows are labeled A E Note that there is no row B There are eight ports in rows A and C and seven in rows D and E The port labeled F is sized for a standard Luer lock connector
18. dance with the intended use described and the written instructions provided in this user guide No license to make or sell products by use of this product is granted to the buyer whether expressly by implication by estoppels or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research please contact NUGEN Technologies Inc 201 Industrial Road Suite 310 San Carlos CA 94070 Phone 888 654 6544 or 650 590 3600 FAX 888 296 6544 or 650 590 3630 Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase provided that the product is handled and stored according to published instructions and that the product is not altered or misused If the product fails to meet these performance standards NuGEN will replace the product free of charge or issue a credit for the purchase price NUGEN s liability under this warranty shall not exceed the purchase price of the product NUGEN shall assume no liabil
19. dd 350 uL Buffer RW1 into the RNeasy mini column and centrifuge for 15 sec onds at 28000 X g 210 000 rpm to wash Discard the flow through 11 Transfer the RNeasy column to a fresh 2 mL collection tube Add 500 uL Buffer RPE with the added ethanol to the RNeasy column 12 Close the tube gently and centrifuge for 15 seconds at gt 8000 X g 210 000 rpm Discard the flow through 13 Add another 500 uL Buffer RPE to the RNeasy column 14 Close the tube gently and centrifuge for 2 minutes at 28000 X g 210 000 rpm Discard the flow through 15 Transfer the RNeasy column to a new 1 5 mL collection tube 16 Pipet 30 50 uL Nuclease free Water D1 green cap directly onto the RNeasy membrane 17 Close the tube gently and centrifuge for 1 minute at gt 8000 X g 210 000 rpm to elute VII Appendix 27 Encore SP Complete Library Systems 18 If yields of greater than 30 ug are expected repeat elution step and collect in the same collection tube DNase Treatment of RNA Post purification Using RNase free DNase and either the RNA Clean and Concentrator 5 Columns or the RNeasy MinElute Columns Note If you are unable to quantify your RNA because the sample is contaminated with DNA we recommend DNase treatment followed by purification 1 On ice mix together 2 5 uL 10X DNase Reaction buffer Roche Cat 04716728001 or USB Cat 78316 with 1 uL rDNase 10 Units Roche Cat 04716728001 or 2 Units USB Cat 78311
20. dge Slowly withdraw the pipette tip from the port performing a final blowout while the tip is within the upper cylinder of the port Note Do NOT add any samples or other reagents to the cartridge at this time Ensure that only Elution Buffer has been loaded Close the lid Select Run on the touch screen menu choose the Mondrian SP Cartridge QC protocol from the list of protocols and then select Next to proceed to the Protocol Information screen Select Next to proceed to the Run Information screen Optional Enter run details on the Run Information screen We recommend record ing the serial number of the cartridge on this screen The cartridge serial number can be found on the front of each cartridge for easy reference Select Next and then select Start Run The Mondrian SP Cartridge QC protocol will take about 15 minutes to complete During this test Elution Buffer droplets will be dispensed from the E5 port and trans ported around the cartridge prior to being returned to port E5 The purpose of this test is to confirm the basic performance of the cartridge IV Protocol 14 Encore SP Complete Library Systems At the end of the protocol the instrument will display the Run Complete screen and one of the following messages MESSAGE MEANING NEXT STEP Mondrian SP Cartridge passed Continue to intended protocol No errors were detected Droplet transport was normal Cart
21. e Encore SP Complete Library Systems on Illumina NGS Platforms The Encore SP Complete Library Systems use a Dedicated Read DR design with a second sequencing primer for multiplex sequencing Figure 2 illustrates the DR multiplex barcode strategy 10 Encore SP Complete Library Systems Ill Planning the Experiment 11 Encore SP Complete Library Systems Figure 2 Dedicated read multiplexing strategy used by the Encore SP Complete Library Systems Dedicated Read Barcode Design Illumina Standard Seq Primer Library Insert Illumina Index Seq Primer a Barcode ELE Flow cell surface The Encore SP Complete Library Systems use the same approach to multiplexing found in the standard Illumina method These libraries should be sequenced using the Illumina protocol for multiplex sequencing The DR Barcode sequences are found in Appendix C of this user guide and must be entered into the Illumina software prior to the analysis C Amplified Library Storage Purified and amplified libraries may be stored at 20 C prior to proceeding with quanti tation cluster generation and sequencing IV Protocol 12 Encore SP Complete Library Systems A Overview The Encore SP Complete Library Systems use a fully automated process on the Mondrian SP Workstation The total time to prepare a purified and enriched library ready for quantitation and sequencing is about 11 hours The Encore SP Complete Library Sys
22. ed to NGS libraries using reagents and adaptors provided as part of the Encore SP Complete Library Systems kit The Encore SP Complete RNA Seq Library Systems have been designed for strand specific expression analysis by incorporation of a nucleotide analog during the second strand cDNA synthesis and subsequent ligation to a pair of double stranded adaptors containing the same analog in one strand After ligation the cDNA strand and adap tor containing the analog are selectively removed Strand Selection leaving only one cDNA strand with both adaptor sequences attached This product is then converted into a sequence ready library by PCR amplification The workflow consists of three steps 1 Hands free automation of the following assay steps on the Mondrian SP Workstation see Figure 1 e RNA sample concentration e cDNA generation e cDNA fragmentation e Library construction End repair Adaptor ligation Strand selection Library amplification e Sample purification 2 Sample collection from the Mondrian SP Cartridge 3 Quantitation cluster formation and sequencing Introduction Figure 1 The three steps of the Encore SP Complete Library Systems workflow Mondrian SP Cartridge Step 2 Make master mix load in reagent ports Filler E1 DTO E E FUILI D1 D2 D3 D4 D5 D D7 Sample Collection AAA r 8 6 7 3 9000 Adaptors Step 1 100 150 ng total RNA Encore SP Comple
23. eeeescersaeesscecsaeeceeesseeesaeesaeeeeaeseneeeeneeeneeees 5 B Additional Equipment Reagents and Labware cccceceseeeeeeeeeeeeeseeneens 9 Ill Planning the Experimenitisccccccccicesccccccccesscecscecsscscecscccxoeseecs cody esuueszesessescoseeees sises 10 As IInput RNA Requirements lt ics 2 sd ccsccssncdesahacscssdtedicndtidsttnassiessenaiessncbeatceesecten 10 B Using the Encore SP Complete Library Systems on lumina NGS Platine cosecessais sve tees eane ents E E 10 C Amplified Library Stora Ge i 2 ccceesscetesseartasdestiessardessnscvsrtessssieertacscsascetabtasteceass 11 IV Protocol sccscscscsssssssssecccasssessssnessasavsassesensessesssssneassescessosccsassveasssseassssvisassocsssvseadss 12 As ON GINIGW acsee ea e E e nencduartes iosenesthedesbaestustenanseans 12 B Protocol Notessa esris inaina ceidngsscbastions auacvtedescheesteantendsteneate 12 C Cartridge Quality Control Check cece eee ee eee cee eeeteeeceeteeeseeteretneeniee 12 D Protocol for Encore SP Complete Library Systems on the Mondrian SPE Ca tg ess c tosgnedeatttatessacetiagsodessandascnecnesdiadeaUoastagessetessentatdeantes 14 V Quantitative and Qualitative Assessment of the Purified Amplified Libraries 24 A OVGIWIOW nserit aas E E E sigs vacks taaes es aah eastb ada he A 24 B Recommendations for the Quantitative Assessment of the Purified Amplified RNA Seq Libraries 0 0 ccc icc cee eeee rene ceeeceeeceeeteseeneeseeeiees 24 VI Technical Suppor
24. entrifuge tubes in a rack Add 15 uL of Nuclease free Water green D1 to each tube Use a 100 or 200 uL pipette set to 20 uL Depress the plunger on the pipette and insert the tip all the way to the bottom of the sample collection port perpendicular to the cartridge so as to make a seal between the cartridge and the pipet tip Maintaining the seal formed between the pipette tip and the bottom of the cartridge release the plunger Immediately lift the pipette slightly off the bottom of the cartridge to release the seal rapidly drawing Filler Fluid and the sample droplet into the pipette tip Examine the pipette tip to ensure that the appropriately sized droplet is sus pended in the Filler Fluid in the pipette tip Repeat steps 2 4 above if the prepared library droplet was not captured the first time If no sample droplet is observed dispense the contents of the pipette tip into the sample collection vessel and repeat the sample collection procedure for the port Note that the droplet occasionally breaks into pieces and becomes more difficult to observe The excess Filler Fluid collected during the procedure will not interfere with downstream molecular processes Continue to the next sample collection port and repeat this process until all eight libraries have been collected and placed in separate tubes Remove the cartridge from the workstation and dispose of as appropriate in labo ratory waste IV Protocol 23 Encore SP Comple
25. er 11 01712 First Strand Buffer Mix 1 of 2 Blue A2 vER 9 01713 First Strand Enzyme Mix 1 of 2 Blue A3 VER 5 S01714 Second Strand Buffer Mix 1 of 2 Yellow B1 ver 7 7 Components Encore SP Complete Library Systems Encore SP Complete DR Multiplex System 9 16 Components and Reagents Part No 8152 continued 8152 8152 8152 8152 8152 PART VIAL NUMBER DESCRIPTION BOX CAP VIAL NUMBER 01715 Second Strand Enzyme Mix 1 of 2 Yellow B2 ver 4 S01717 Fragmentation Enzyme Mix 1 of 2 Orange F2 veR 3 S01716 Fragmentation Buffer Mix 1 of 2 Orange F1 ver 3 01627 End Repair Buffer Mix 1 of 2 Blue ER1 ver 5 01510 End Repair Enzyme Mix 1 of 2 Blue ER2 ver 4 01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 01730 DR Multiplex Ligation Adaptor 1 of 2 Yellow L2V10DR BC9 01731 Mixes 9 16 L2V10DR BC10 01732 L2V10DR BC11 01733 L2V10DR BC12 01734 L2V10DR BC13 01735 L2V10DR BC14 01736 L2V10DR BC15 S01737 L2V10DR BC16 S01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 S01718 Strand Selection Buffer Mix 1 of 2 Purple SS1 veR 2 01512 Strand Selection Enzyme Mix 1 of 2 Purple SS2 01738 Strand Selection Enzyme Mix II 1 of 2 Purple SS4 501807 oneal 1 of 2 Red SS3 P1 Amplification Buffer Mix S01765 Amplification Primer Mix 1of2 Red P2 ver 9 S01764 Amplification Enzyme Mix 1 of 2 Red P3 ver 2 01001 Nuclease Free Water 1 of 2 Green D1 PO1208 Mondrian SP Cartridges x4 2 o
26. f 2 N A N A Encore SP Complete Library PO1212 Systems Cartridge Loading 2 of 2 N A N A Guide x4 8 Components Encore SP Complete Library Systems Encore SP Complete DR Multiplex System 9 16 Components and Reagents Part No 8152 continued ee 8152 8152 wg 8152 NUMBER DESCRIPTION BOX CAP VIAL NUMBER S01719 Mondrian SP Filler Fluid x4 2of2 N A N A 01587 sampe Concentration 2of2 Clear N A Solution S01588 Bead Binding Solution 2of2 Clear N A 01589 Bead Wash Solution 2 of 2 Clear N A S 01590 Elution Buffer 2 of 2 Clear N A 01825 Bead Additive hiippec Clear N A separately 501698 Agencourt RNAClean XP Shipped Clear N A Beads separately ll Components 9 Encore SP Complete Library Systems B Additional Equipment Reagents and Labware Required e Eq e Re e Su Materials uipment Mondrian SP Workstation Part No 8100 Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic analysis of nucleic acids Microcentrifuge for individual 0 5 mL and 0 2 mL tubes 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer agents sopropyl alcohol Low EDTA TE buffer pH 8 0 Affymetrix Cat 75793 pplies and Labware Nuclease free pipette tips 0 5 mL and 0 2 mL RNase free microcentrifuge tubes Disposable gloves Lint free wipes such as Kimwipes or Berkshire Super PolX 1200 Wipers VWR Cat 21
27. hlighted with a white rim outlined in black O 11 Load 8 uL Ligation Master Mix tube D5 into D5 Port D5 is high D5 lighted with an orange rim O 12 Load 8 uL End Repair Master Mix tube D6 into D Port D is high D lighted with a green rim O 13 Load 8 uL Fragmentation Master Mix tube D7 into D7 Port D7 is D7 highlighted with a dark blue rim O 14 Load 50 uL of sample mix from Step 1 above into 1 S8 Ensure that the 10 minute incubation step described above is performed prior to loading onto the cartridge Sample input ports are highlighted in red Sample Input C0066000 IV Protocol 22 Encore SP Complete Library Systems 15 If the cartridge is not already inserted into the Mondrian SP Workstation deck insert the cartridge into the deck and pull the locking lever forward to engage the cartridge with the control electronics and close the lid Mondrian SP Workstation Initialization Instructions 1 If the workstation is not already ON locate the workstation ON OFF switch at the back of the workstation and turn switch to the ON position Press the On button on the front of the workstation Select Run from the Main Menu touch screen select the Encore SP Complete protocol from the Protocol Selection menu and press Next Follow the instruc tions on the screen to begin the run Library Collection from the Mondrian SP Cartridge Place eight 0 2 mL or 0 5 mL microc
28. ilar percentages can be achieved with chicken and zebrafish total RNA Use with lower eukary otes such as D melanogaster or C elegans may result in higher rRNA read percentages Will this system capture small RNA species The Encore SP Complete Library Systems were designed to create libraries of intact mRNA species We have not tested any modifications to Encore SP Complete Library Systems protocol that enable the capture of smaller RNA species Can contaminating genomic DNA interfere with the Encore SP Complete Library Systems performance Yes When using purified total RNA samples contaminating genomic DNA may be incorporated into libraries For this reason we recommend DNase treatment during RNA purification For an explanation of DNase requirements see Section IIl A 4 For guidelines on DNase treatment of RNA samples refer to Appendix A Is this system compatible with target enrichment strategies Yes The Encore SP Complete Library Systems are compatible with down stream target enrichment Specific blocking sequences may be necessary depending upon the selection technology Please contact NuGEN Technical Support for additional information How should measure my amplified cDNA product yield Can I use an Agilent Bioanalyzer to evaluate the product Yes Refer to Section VI of the user guide for guidelines on Quantitative and Qualitative Assessment Where can safely stop in the protocol Purified and amplified libra
29. ired end assay The workflow generates libraries with an average insert size of 200 bases This length corresponds to the expected distance between the 5 most and 3 most coordinates of paired end reads Do I need to perform a separate sequencing read for the multiplex adaptors Yes A separate sequencing read is required to sequence the multiplex adap tors The Encore SP Complete Library Systems barcoding strategy requires a separate dedicated sequencing read to identify the barcode VII Appendix Q25 How many bases do the Encore SP Complete Library Systems multiplex adaptors add to the library The adaptors add 122 bp to the library inserts generated from the Encore SP Complete Library Systems Q26 What is the expected yield of the enriched DNA library using the Encore SP Complete Library Systems The expected yield is between 100 300 ng depending on the quality and quantity of the input RNA This amount is a large excess over the amount of DNA required for use on the cBot or Cluster Station Q27 Can use standard alignment algorithms to analyze strand specific sequencing data Yes Strand specific reads can be processed and mapped to reference sequences using the same methods used for other RNA Seq libraries Of the reads from Encore SP Complete Library Systems libraries gt 90 will align in the sense strand orientation relative to the RNA template F Update History This document the Enc
30. ity for direct indirect consequential or incidental damages arising from the use results of use or inability to use its products NUGEN reserves the right to change alter or modify any product to enhance its performance and design NuGEN s products are developed designed and sold FOR RESEARCH USE ONLY This product is not to be used for diagnostic or therapeutic purposes nor is it to be administered to humans or animals Except as expressly set forth herein no right to modify reverse engineer distribute offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NUGEN product The buyer agrees to use NUGEN products accompanying the product insert in accordance with the intended use and the written instructions provided Table of Contents Contents Ik MNEFOGUCHION ss ccsssssscsssessssssessssvecsescsssssssstacssessessssssssesnesasovsaasossusensasssssvscadssesseasssssess 1 As Backgrounder e E E sands A E E E 1 B Performance Specifications xcssic cacessncveuseacisessuccssecchdesvncsavbenschcvavucenslechevachens 3 C OwalityCOmerol a ere E ar e Ea EE 3 D Storage and Stability cicsccasicsacssiececcucesnncssevidees conse ae aas 3 E Material Safety Data Sheet MSDS 0 cecceeceeeceeeeeeeeeeeeeeceeeeeeeeeeeeeaeeneeeeeeees 4 Ii COMPONCNES lt 5 c scssesccsscctessscessatesascucssavsnvesuesdsvesassecsesvesassusaedsdsvasstacisesvaseseccessoasesiess 5 A Reagents Provided cceecceesceseceeences
31. ix Mix by pipetting and spin COMPONENT VIAL CAP VOLUME down the master mix ced di Seminars End Repair Buffer Mix ER1 ver 5 Blue 9 0 uL End Repair Enzyme Mix ER2 ver 4 Blue 1 0 uL Total volume 10 0 pL Prepare Fragmentation Master Mix 1 Thaw the Fragmentation Buffer Mix orange F1 ver 3 at room temp and vortex to mix well Spin and place the Fragmentation Enzyme Mix orange F2 ver 3 on ice 2 Prepare the Fragmentation Master Mix in a 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 9 Label the tube D7 Table 9 Fragmentation Master Mix Mix by pipetting and spin COMPONENT VIAL CAP VOLUME down the master mix briefly Place on ice Use immediately Fragmentation Buffer Mix F1 ver 3 Orange 8 7 uL 17 Encore SP Complete Library Systems Fragmentation Enzyme Mix F2 ver 3 Orange 1 3 pL Total volume 10 0 pL IV Protocol Mix by pipetting and spin down the master mix 18 briefly Place on ice Use immediately Encore SP Complete Library Systems Prepare Strand Selection II Library Amplification Master Mix 1 Thaw the Strand Selection II Amplification Buffer Mix red SS3 P1 and Amplification Primer Mix red P2 ver 9 at room temp and vortex to mix well Spin and place the Strand Selection Enzyme Mix II purple SS4 and the Amplification Enzyme Mix red P3 ver 2 on ice Prepare the Strand Selection II Library Amplification Master Mix in a 0 5 mL
32. lex up to 16 samples B Performance Specifications The Encore SP Complete Library Systems are designed to generate DNA libraries suitable for either single read or paired end sequencing on Illumina Genome Analyzer IIx lle GAIIx MiSeq HiScan SQ or HiSeq 2000 2500 NGS platforms This simple hands free and robust system is capable of starting with 100 ng of total RNA to gener ate libraries ready for quantitation and cluster formation in about 11 hours C Quality Control Every lot of the Encore SP Complete Library Systems undergoes functional testing to confirm that the products meet the specifications for library generation performance We recommend the use of control samples when beginning experiments and or using a new source of samples For RNA based experiments such as RNA Seq we recom mend using the Microarray Quality Control MAQC reference samples A and B D Storage and Stability The Encore SP Complete Library Systems reagents are shipped in two boxes Box 1 is shipped on dry ice and upon receipt should be stored at 20 C on an internal shelf of a freezer without a defrost cycle Box 2 is shipped at room temperature but contains components with multiple storage temperature requirements and should be unpacked immediately upon receipt Introduction e Vials labeled Agencourt RNAClean XP Beads clear cap and Bead Additive S01825 should be removed from the top of the Box 2 shipping carton upon delivery
33. ling plate you may gently press down on them to allow the cartridge to move forward The heater bars and cooling unit are designed to be slightly flexible in order to engage the cartridge However do not press too hard on the heater bars or Peltier unit as they may become misaligned 31 Encore SP Complete Library Systems VII Appendix 32 Encore SP Complete Library Systems C Confirm that the cartridge is fully inserted into the deck When the cartridge is properly inserted the electrodes are no longer visible and the three guide arrows etched into the front of the deck should be flush with the edge of the cartridge Pull the locking lever forward as illustrated above to engage the control electronics and lock the cartridge into place Note Once a run has started do not move the locking lever as this will result in an unrecoverable interruption of the running protocol The Mondrian SP Workstation will not run with the lever in the up position Cleaning the Workstation Contact Pins The interface pins on the workstation cartridge interface may become dirty and cause performance issues If this happens you will need to clean the pins Materials e Lint free wipes such as Kimwipes or Super PolX 1200 Wipers Do not use cotton or any material that may leave particles behind e Isopropyl alcohol e Canned air Procedure 1 2 3 4 5 Turn the workstation off and unplug from the power source Soak the wipe in is
34. m contain a SPIA based amplification No The cDNA is generated with selective primers but without SPIA based amplification don t want to do multiplex sequencing but the Encore SP Complete Library Systems are available only with multiplex barcodes How can I perform non multiplexed sequencing The Encore SP Complete Library Systems kit can be used for non multiplexed sequencing If you do not wish to perform multiplex sequencing follow the protocol as outlined in Section IV D but do not combine samples prior to sequencing What equipment is required or will be useful when using the Encore SP Complete Library Systems A comprehensive list of required and recommended equipment can be found in Section III B of this user guide Can I use the Encore SP Complete Library Systems with RNA from any organism This system has been designed specifically for higher vertebrates such as human mouse rat frog zebrafish and chicken Performance with RNA from other organisms may vary and should be empirically tested prior to beginning a project Do I need to use high quality total RNA The Encore SP Complete Library Systems are designed to work with puri fied total RNA When using purified total RNA samples should be of high molecular weight with little or no evidence of degradation While it is impos sible to guarantee the highest levels of performance when using RNA of lower quality this system should allow the successful analysis of
35. mercially available system that does not require organic solvents If using a method such as Trizol we recommend column purification after isolation One measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm The A260 A280 ratio for RNA samples should be in excess of 1 8 3 RNA Integrity RNA samples of high molecular weight with little or no evidence of degradation will perform very well with this product In many samples RNA integrity can be determined using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano LabChip or RNA 6000 Pico LabChip These instruments provide sensitive and rapid ways of confirming RNA integrity prior to processing While it is impossible to guarantee satisfactory results with all degraded samples this system can work with many samples that are moderately degraded 4 DNase Treatment We highly recommend using DNase treated RNA with this system The presence of genomic DNA in the RNA sample may have adverse effects on downstream ana lytical platforms If the total RNA sample contains a significant amount of genomic DNA it may be difficult to accurately quantify the true RNA concentration The RNA input quantity may therefore be overestimated based on an absorbance measurement Since it is important that RNA input be between 100 ng and 150 ng we recommend using a DNase treatment that will remove genomic DNA during RNA purification See Appendix A for recommendations and protocols B Using th
36. nto the port marked Filler Fluid on the cartridge while gently twisting the tube in a clockwise direction to secure the Filler Fluid tube on the cartridge Remove the rubber stopper from the bottle of SP Filler Fluid Slowly pour the SP Filler Fluid into the Filler Fluid tube Be careful not to pour too quickly as this may introduce bubbles into the fluid Air bubbles entering the cartridge may interfere with droplet movement During the cartridge filling pro cess you will begin to see the Filler Fluid rising in the sample and reagent ports You should see Filler Fluid in all sample and reagent ports by the end of the filling process Once the entire volume of Filler Fluid has been delivered carefully remove the Filler Fluid tube from the cartridge VII Appendix Cartridge Insertion 1 Raise the lid of the Mondrian SP Workstation to reveal the cartridge deck 2 With the cartridge lever in the up position as shown above hold the cartridge level by the sides with the ports facing up and the gold colored electrodes pointing towards the workstation Carefully insert the cartridge into the cartridge deck flush with the surface of the deck allowing the side rails to guide it into place 3 Ifyou encounter any resistance to the cartridge moving into the deck inspect for foreign objects that may be impeding the cartridge and remove them If the cartridge appears to be catching on the edge of the heater bars or Peltier coo
37. o the additional length conferred by the adaptors and primers ligated onto the initial 200 bp fragment The distribution pattern of the amplified library inserts depends upon the initial frag ment size used to construct the library Following the Encore SP Complete protocol effectively eliminates adaptor dimer formation Any deviation from the protocol may result in dimer formation which will appear as low molecular weight spikes of approxi mately 120 bp on the Bioanalyzer trace Figure 4 Library Insert Distribution Distribution of amplified purified Encore SP Complete Library System library inserts on a Bioanalyzer HS DNA Chip 1000 Library MC31C_1 was made from 100 ng human brain total RNA and library MC31C_5 was made from 100 ng Universal Human Reference total RNA MC31C_1 MC31C_5 Fu 35 150 300 500 1000 10380 bp 35 150 300 500 10380 fl 2 Validate the library as described in Illumina user guides for DNA library construc tion e g Genomic DNA Sample Prep Manual Cat FC 102 1001 VI Technical Support 25 Encore SP Complete Library Systems For help with any of our products please contact NUGEN Technical Support at 650 590 3674 direct or 888 654 6544 option 2 toll free U S only You may also send faxes to 888 296 6544 toll free or email techserv nugen com In Europe contact NUGEN at 31 0 135780215 Phone or 31 0 135780216 Fax or email europe nugen com In all other locations contact your NU
38. opropyl alcohol Firmly rub all pins with the wipe Wait 2 minutes Blow the area dry with canned air VII Appendix 33 Encore SP Complete Library Systems 6 Plug the workstation back in and turn it on Note We recommend cleaning the pins once a week after any prolonged storage of the workstation after a Filler Fluid spill or if the Mondrian SP Cartridge QC proto col returns a Mondrian SP Cartridge Failed or a Mondrian SP Cartridge Status Undetermined message D Sequences of the DR Barcodes in the Multiplexed Reactions Barcode sequences for adaptors used in the Encore SP Complete Library Systems can be found in Tables 12 and 13 These six nucleotide barcode adaptor sequences must be entered into the Illumina Sequencing System prior to parsing of the data You may combine between 2 and 8 barcoded adaptors to allow for a range of multiplex sequencing Table 12 Barcode sequences for dedicated read DR adaptors used in Encore SP Complete Multiplex System 1 8 Part No 8151 Barcode Pane 6 nt Barcode Adaptor Pairing Barcode Adaptor Ligation Pairing for 3 plex 3 Sequence as Read for 2 plex Adaptor Mix 3 Through 8 plex by the Sequencer Multiplex x Sequencing Multiplex Sequencing L2V10DR BC1 AACCAG Duplex Set 1 L2V10DR BC2 TGGTGA LOR Be AGTGAG One of the duplex sets L2V10DR BC4 GCACTA Duplex Set 2 from the column to the i left must be used in L2V10DR BC5 ACCTCA combination with
39. ore SP Complete Library System User Guide M01331 v4 is an update to address the following topics Description Section Page s Updated volumes in Table11 IV D 17 Updated mixing directions for Bead Master Mix IV D 19 NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 109 For our international distributors contact San Carlos CA 94070 USA 9350 AC Leek information visit our website Toll Free Tel 888 654 6544 The Netherlands U Toll Free Fax 888 296 6544 Tel 31 13 5780215 Peer imagine more from lesse custserv nugen com Fax 31 13 5780216 techserv nugen com europe nugen com 2013 2014 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugen com NuGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners For research use only M01331 v4
40. other NuGEN kits and systems should not be used with the Encore SP Complete Library Systems C Cartridge Quality Control Check The Mondrian SP Cartridge QC protocol confirms the basic functionality of the Mondrian SP Cartridge prior to use We recommend running this protocol with each cartridge prior to preparing or adding samples and reagents 1 We recommend cleaning the contact pins on the deck of the workstation prior to running the Mondrian SP Cartridge QC Protocol The cleaning procedure is detailed in Appendix C 2 On the bench top fill the cartridge with Filler Fluid via the Filler Fluid port accord ing to the instructions in Appendix B IV Protocol 13 Encore SP Complete Library Systems 10 11 Carefully transport the cartridge to the workstation and insert it into the deck Pull the cartridge lever of the Mondrian SP Workstation forward to the locked position Place the Cartridge Loading Guide on the cartridge or use the guide as a refer ence to identify the correct port for loading Elution Buffer Pipette 50 uL of Elution Buffer into port E5 of the Mondrian SP Cartridge Insert the pipette tip into the port all the way to the bottom of the cartridge When the tip contacts the bottom withdraw the pipette tip slightly to allow space for dis pensing Slowly depress the plunger to dispense the reagent but do not depress the plunger completely blow out as this could introduce bubbles into the car tri
41. ridge is ready to runa protocol Press OK on the Run Complete screen to return to the main menu Proceed to the intended protocol Mondrian SP Cartridge failed Remove cartridge from instrument deck and set aside prior to contacting NuGEN Technical Support A problem was detected with droplet trans port within the cartridge Press OK on the Run Complete screen to return to the main menu Carefully remove the cartridge from the deck of the workstation setting aside the cartridge for possible return to NuGEN Begin with a new cartridge and contact NuGEN Technical Support to request a replacement for the failed cartridge D Protocol for Encore SP Complete Library Systems on the Mondrian SP Cartridge 1 Sample Solution Mix Preparation Prepare the Sample Solution Mix for loading onto the cartridge This is done on a per sample basis and not as a master mix You must prepare and process no fewer than eight samples on each cartridge Table 3 Sample Solution Mix volumes given are for one sample COMPONENT VOLUME 100 to 150 ng of total RNA 23 5 pL Agencourt RNAClean XP beads 4 0 uL Sample Concentration Solution 27 5 pL Total volume 55 pL Ensure the Agencourt RNAClean XP beads are at room temperature and completely resuspended prior to use Keep the Agencourt RNAClean XP beads at room temp after IV Protocol Mix by pipetting and spin down the master mix
42. ries collected from the Mondrian SP Workstation can be placed in short term storage at 20 C prior to proceeding with quanti tation cluster generation and sequencing Does NuGEN provide reagents for performing the fragmentation step of the protocol Yes the Encore SP Complete Library Systems provide the cDNA fragmenta tion reagents as part of the kit VII Appendix 37 Encore SP Complete Library Systems Q18 Q19 Q20 Q21 Q22 Q23 Q24 Can I use alternative fragmentation methods such Covaris shearing instead of using the fragmentation reagents provided in the kit NuGEN does not support or recommend the use of alternative fragmenta tion methods such as Covaris shearing of RNA in lieu of the fragmentation reagents provided in the Encore SP Complete Library Systems Using alterna tive fragmentation methods will impact the performance of the system and invalidate the warranty on the product How much material should I load into the cBot Please follow manufacturer s recommendations for library QC quantitation balancing and loading of the amplified library on the cBot Do the Encore SP Complete Library Systems work with the Illumina Cluster Station predecessor to the cBot instrument Yes The systems are also compatible with the Illumina Cluster Station What kind of error correction is used to minimize the impact of sequenc ing errors in the barcodes Each of the DR Barcode sequences shown in T
43. te DR Multiplex Systems Single use only z Sie Encore SP Complete System reagents Sample Input T 2 7 Mondrian SP Workstation First strand OO performs the cDNA following steps in 11 hrs J Second strand cDNA Fragmentation acces O O O0 it AT J et ssapors ana igote J e VED NNUOIUOUUGR0UE library amplification J Fu 500 Steps 4 5 Library quantitation dL NC take place off the 35 150 300 500 1000 10380 bp Mondrian SP Workstation End repair Strand selection Cluster formation and sequencing 2 Encore SP Complete Library Systems Introduction This product contains components with multiple 3 storage temperatures Encore SP Complete Library Systems Starting with 100 150 ng of total RNA the Encore SP Complete protocol can be completed in approximately 11 hours and yields libraries ready for quantitation cluster formation and either single read or paired end sequencing NuGEN offers a two configurations of the Encore SP Complete Library Systems Encore SP Complete DR Multiplex System 1 8 Part No 8151 and Encore SP Complete DR Multiplex System 9 16 Part No 8152 Each kit provides eight unique dedicated read adaptors to prepare libraries for multiplex sequencing using a dedi cated read design strategy with a second sequencing primer Either kit may be used for up to 8 plex sequencing or the two kits may be used together to multip
44. te Library Systems 10 Cap the library droplet containing PCR tubes and vortex briefly to mix Spin down briefly to bring the aqueous phase to the bottom of the tube The Filler Fluid oil should remain as a separate layer above the aqueous phase Set a 20 uL pipette to 18 uL and remove and discard the Filler Fluid oil layer A very thin layer of Filler Fluid oil may remain in the tube on top of the aqueous phase If the tube contains more than 20 uL of oil repeat this oil removal step until little or no oil remains visible in the tube The liquid remaining in the tubes contains the purified and amplified RNA Seq library Store the library at 20 C or proceed immediately to Section VI Quantitative and Qualitative Assessment of the Purified Amplified Libraries V Quantitative and Qualitative Assessment of the Purified Amplified Libraries 24 Encore SP Complete Library Systems A Overview The Quantitative and Qualitative Assessment is used to confirm the average size of the library inserts and calculate library concentration This information is required prior to loading the cluster generation workstation B Recommendations for the Quantitative Assessment of the Purified Amplified RNA Seq Libraries 1 Runa 1 pL aliquot of the purified amplified libraries on the Bioanalyzer HS DNA Chip 1000 The typical distribution of 200 bp inserts is shown in Figure 4 Note that the actual size of the libraries appears closer to 300 bp due t
45. tems are sold only with DR barcodes However these barcodes may also be used for non multiplex sequencing B Protocol Notes The Encore SP Complete Library Systems are designed and intended for pro cessing eight samples at a time Do not attempt to prepare smaller volume mas ter mixes or process fewer than eight samples using the Encore SP Complete Library Systems We recommend the routine use of a positive control RNA Especially the first time you set up a reaction using a positive control will allow you to establish a performance baseline Use the Nuclease free Water provided with the kit D1 green or an alternate source of nuclease free water We do not recommend the use of DEPC treated water with this protocol Thaw the components used in each step and immediately place them on ice Always keep thawed reagents and reaction tubes on ice unless otherwise instructed After thawing and mixing buffer mixes if any precipitate is observed re dissolve the precipitate completely prior to use You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing Do not warm any enzyme or primer mixes When placing small amounts of reagents into the reaction mix pipet up and down several times to ensure complete transfer When instructed to pipet mix a solution or master mix gently aspirate and dis pense a volume that is at least half the total volume of the solution master mix Components and reagents from
46. ten iaeiiai raean KKEL KO EEEE ESSERNE 25 MILAPpEndiXK sssini e a a EE E E E EEEE 26 A DNase Treatment oft RNA risintia t E Eae ai 26 B Mondrian SP Cartridge Handling s ssssssssssssssissssssisisisrisistsristnsisrinrisinsrsna 28 C Cleaning the Workstation Contact Pins cccceccsseeseeseeceseeseeseeseneeerenseneeaeens 32 D Sequences of the DR Barcodes in the Multiplexed Reactions ee 33 E Frequently Asked Questions FAQS ecsssusenaca ni 35 E Update HETON iarria onie E EE EEE OE 38 1 Introduction Encore SP Complete Library Systems A Background The Encore SP Complete Library Systems are complete reagent cartridge and pro tocol kits for the simple automation of RNA Seq library preparation protocols using the Mondrian SP Workstation These systems enable RNA Seq library construction using as little as 100 ng of total RNA The core technology used in this product enriches for coding and regulatory transcripts in NGS libraries during cDNA synthesis and can be applied to transcriptomes from a broad range of higher eukaryotes The cDNA synthe sis is carried out using proprietary primers to create double stranded cDNA that retains strand specific expression information The resulting sequencing reads can be aligned to the strand from which the RNA originated enabling detection of both sense and antisense expression No dedicated steps are required to reduce rRNA levels in the final NGS library The resulting cDNA is convert
47. through which the Filler Fluid is added to the cartridge The cartridge electrodes are visible as rows of small gold squares at the top of the cartridge E Reagents and master mixes F Filler Fluid port D Reagents and master mixes C Collection of final purified libraries A Adaptor ports Bubble trap ports nothing is added or removed via these ports Sample input ports VII Appendix The SP Filler Fluid has been degassed and pack 30 aged in a sealed single use vial Do not open the vial until ready for use Encore SP Complete Library Systems Filling the Cartridge SP Cartridges must be filled with the provided Filler Fluid prior to loading with reagents This process should be carried out on the bench top 1 Use scissors to cut open the Mondrian SP Cartridge pouch Remove the Mondrian SP Cartridge Note Do not attempt to tear open the pouch as this may damage the cartridge Lay the cartridge on a level bench top and place the appropriate SP Library Systems Cartridge Loading Guide on the cartridge Remove the empty Filler Fluid tube and the SP Filler Fluid bottle from the NuGEN SP Library System kit Carefully grasp the tab on the red aluminium seal of the SP Filler Fluid bottle and pull up and back to remove the seal Remove the top and bottom caps from the empty Filler Fluid tube Attach the empty Filler Fluid tube to the SP Cartridge by inserting the tip of the Filler Fluid tube i
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