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TruSeq RNA Access Library Prep Guide - Support

1. Part 15049525 Rev B Supporting Information Introduction 3 n oaie at dotari nee tarta 66 ACTOS oe ede col bent isa 67 Kit CONIENIS e n oaie ee esc Se iati ete deh SAS stare cia os qa e eos ea e DA 70 Consumables and Equipment aaa 76 TruSeq RNA Access Library Prep Kit Indexed Adapter Sequences 82 prs re pier Wk Commis NS QU E 3 SS ZA AS nn el nasa x Z nal m a ran ct A i E Or AAT ce caca rea pegas cert TACTO paa RR maA Ea na rz 9 4 P SEM ae e or ES y pa D Ee 7 Se oz So TruSeq RNA Access Library Prep Guide 6 5 v xipueddw Supporting Information Introduction The protocols described in this guide assume that you have reviewed the contents of this appendix confirmed your kit contents and obtained all of the requisite consumables and equipment 6 6 Part 15049525 Rev B Acronyms Table 4 TruSeq RNA Access Library Prep Acronyms Acronym ALP ATL CAP GE cDNA CEX CPP Cg DEPC DFP ds cDNA dsDNA DV EET EPH EPM ET2 EUC TruSeq RNA Access Library Prep Guide Definition Adapter Ligation Plate A Tailing Mix Clean Up ALP Plate cDNA Clean Up Plate Complementary DNA Coding Exome Oligos Clean Up PCR Plate Capture Target Buffer 3 Diethylpyrocarbonate Depleted RNA Fragmentation Plate Double Stranded Complimentary DNA Double stranded DNA Fragment distribution value Enrichment Elution Buffer 1 Elute Prime Fragmen
2. Lower amounts might result in inefficient ligation and low yield The protocol has been tested using 10 ng of high quality universal human reference total RNA as input Use of RNA from other tissues or qualities might require further optimization regarding the initial input amount It is important to know the quality of the RNA starting material The fragmentation conditions were optimized for high quality RNA The following figure shows a Universal Human Reference UHR starting RNA Bioanalyzer trace Figure 1 Starting RNA Bioanalyzer Trace Degraded or FEPE RNAs are shorter than full length RNA RNA that has DNA contamination results in an underestimation of the amount of RNA used TruSeq RNA Access Library Prep Guide 3 suonepueuuulooeH Indu vNH Overview If starting with FEPE RNA the sample input amount is based on sample quality Illumina recommends using the percentage of RNA fragments gt 200 nt fragment distribution value DV as a reliable determinant of FFPE RNA quality Table 1 FFPE RNA Input Recommendations Quality DV Input Requirement Per Reaction High gt 70 20 ng Medium 50 70 20 40 ng Low 30 50 40 100 ng Too Degraded lt 30 Not recommended For successful library prep Illumina recommends using an RNA isolation method that includes a reverse crosslinking step and DNasel treatment such as the QIAGEN RNeasy FFPE Kit or QIAGEN AllPrep DNA RNA FFPE Kit Illumina determines FFPE RNA concentration by Nanod
3. When indexing libraries using adapter index tubes Illumina recommends arranging samples that are going to be combined into a common poolin the same row Also include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol Stop Ligation Buffer t NOTE i Do not remove the Ligation Mix tube from 25 C to 15 C storage until instructed to do so in the procedures Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the AMPure XP beads from storage and let stand for at least 30 minutes to bring them to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 7 for information on how to access TruSeq RNA Access Library Prep Best Practices on the Illumina website Pre heat the microheating system 1 to 30 C Label a new 96 well MIDI plate CAP Clean Up ALP Plate with a smudge resistant pen Label a new 96 well HSP plate PCR Polymerase Chain Reaction Plate with a smudge resistant pen Centrifuge the thawed RNA Adapter tubes at 600 x g for 5 seconds Immediately before use remove the Ligation Mix tube from 25 C to 15 C storage Remove the adhesive seal from the ALP plate Add 2 5 ul Resuspension Buffer to each well of the ALP plate TruSeq RNA Access Library Prep Guide P T sJo1depy e1e6r Protocol 5 Add 2 5 ul Ligation Mix to each
4. 3ATAACAGTAACACACT TCTGTTAACCTIAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTTGATCCACTGAT TCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACG TACCG TA ATCAAT TGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGTTAACCTTAAGAT TACTT GATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAACGACGA SAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG AT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACA CCA CCTTAAGATTACTT C A VT TGAGACTAAATAT TAACG A AA CGAACTTCTGTTAA 3CIACCGTGCAACGAAAATAACCT TAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACG TATCAAT TGAGACTAAGCTACCGT GCAACGACGAAAAGAAT GAT A ae wise O GATE GAC SATI CAE A die EQUI USE M AOV TA PIE A YE a Sg VER AREIS TS S EUIS 3ATAACAGTAACACACT TCTGTTAACCT T Su AES E S pie p RH SEES TCAATTGAGACT m Uu CGACGAAAAGAATGATAACAGTAACACAC TTC TGT I CCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAA 3aATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCG TCTTCTGT TAACCTTAAGAT TACTTGATCCACTGAT TCAACG GTACCGT eU E MAU a ae E vr aces ATCAATTG
5. Remove the ALP plate from 25 C to 15 C storage if it was stored at the conclusion of Clean Up DEP on page 22 Let it thaw at room temperature Centrifuge the thawed ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Pre heat two microheating systems system 1 to 37 C and system 2 to 70 C Centrifuge the thawed A Tailing Mix tube at 600 x g for 5 seconds Add 2 5 ul Resuspension Buffer to each well of the ALP plate Add 12 5 ul thawed A Tailing Mix to each well of the ALP plate Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes Centrifuge the ALP plate at 280 x g for 1 minute Return the A Tailing Mix tube to 25 C to 15 C storage Incubate 1 ALP 1 Place the sealed ALP plate on the pre heated microheating system 1 Close the lid and incubate at 37 C for 30 minutes Immediately after the 37 C incubation remove the ALP plate from system 1 and place the plate on the pre heated microheating system 2 Close the lid and incubate at 70 C for 5 minutes Set the microheating system 1 to 30 C in preparation for Ligate Adapters Immediately remove the ALP plate from the microheating system 2 and place the plate on ice for 1 minute Proceed immediately to Ligate Adapters on page 26 TruSeq RNA Access Library Prep Guide 2 D Spu3 e aje huspy Protocol 26 Ligate Adapters This
6. This protocol explains how to convert total RNA into a library of template molecules of known strand origin then capture the coding regions of the transcriptome using the reagents provided in Illumina TruSeq RNA Access Library Prep kits The resulting library is suitable for subsequent cluster generation and sequencing The RNA is fragmented into small pieces using divalent cations under elevated temperature cDNA is generated from the cleaved RNA fragments using random priming during first and second strand synthesis and sequencing adapters are ligated to the resulting double stranded cDNA fragments The coding regions of the transcriptome are then captured from this library using sequence specific probes to create the final library This library prep protocol offers High data quality even from degraded or FFPE derived RNA samples Input requirement as low as 10 ng for fresh frozen samples and 20 ng for FFPE samples Uniform capture of the coding transcriptome reducing sequencing requirement while maintaining discovery power Up to 24 unique indexes and 4 plex pre enrichment pooling for the most efficient use of your sequencing read budget Strand information on RNA transcripts High throughput automation friendly procedures 2 Part 15049525 Rev B RNA Input Recommendations It is important to follow the TruSeq RNA Access Library Prep input recommendations Total RNA Input This protocol is optimized for 10 100 ng of human total RNA
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8. Adapter Index 21 NA Adapter Index 22 NA Adapter Index 23 NA Adapter Index 25 NA Adapter Index 27 NA Adapter Index 1 NA Adapter Index 3 NA Adapter Index 8 NA Adapter Index 9 NA Adapter Index 10 NA Adapter Index 11 AA AAA ADRAAABWRAA 73 S1U91UO2 Uy Capture Reagents Box 3 Store at 2 C to 8 C This box is shipped on refrigerated gel packs As soon as you receive your kit store the components at 2 C to 8 C Figure 11 TruSeq RNA Access Capture Reagents 48 Samples Box 3 part 4 15052313 OOO 000 e Slot Reagent Part 4 Description 13 SMB 15015927 Streptavidin Magnetic Beads 2 ER 15013008 Elute Target Buffer 2 Supporting Information Ki A Partit 15049525 Rev B Coding Transcriptome Box 4 Store at 25 C to 15 C This box is shipped on dry ice As soon as you receive your kit store the components at 25 C to 15 C Figure 12 TruSeq RNA Access Coding Transcriptome 48 Samples Box 4 part 15052314 Slot Reagent Part Description 1 3 Coding Exome Oligos Capture Target Buffer 3 5 6 Enrichment Elution Buffer 1 7 Enhanced PCR Mix 2N NaOH 9 Enrichment Wash Solution TruSeq RNA Access Library Prep Guide v 5 S USJUO 1M Supporting Information Consumables and Equipment 76 Check to make sure that you have all of the necessary user supplied consumables and equipment before starting the TruSeq RNA Access Library Prep protocol A NOTE The TruSeq RNA Access L
9. Illumina website at www illumina com msds Preparation Remove one tube of First Strand Synthesis Act D Mix from 25 C to 15 C storage and thaw it at room temperature TruSeq RNA Access Library Prep Guide 1 rj VNQ pues 18114 9ZIS9SYJUA S Protocol Add FSA 18 Pre program the thermal cycler with the following program and save as Synthesize 1st Strand Choose the pre heat lid option and set to 100 C 25 C for 10 minutes 42 C for 15 minutes 70 C for 15 minutes Hold at 4 C Make sure that the microplate shaker is properly calibrated to 1000 rpm using a stroboscope y NOTE The First Strand Synthesis Mix Act D with SuperScript II added is stable to additional freeze thaw cycles and can be used for subsequent experiments If more than six freeze thaw cycles are anticipated divide the First Strand Synthesis Mix Act D and SuperScript II mix into smaller aliquots and store at 25 C to 15 C Remove the adhesive seal from the DFP plate Centrifuge the thawed First Strand Synthesis Mix Act D tube at 600 x g for 5 seconds Add 50 ul SuperScript II to the First Strand Synthesis Act D Mix tube Mix gently but thoroughly and centrifuge briefly If you are not using the entire contents of the First Strand Synthesis Act D Mix tube add SuperScript II at a ratio of 1 ul SuperScript II for each 9 ul First Strand Synthesis Act D Mix Label the First Strand Synthesis Mix Act D tube to indicate that the SuperScript II has been added Add 8
10. PCR enriches for fragments that have adapters ligated on both ends Fragments with only one or no adapters on their ends are by products of inefficiencies in the ligation reaction Neither species can be used to make clusters Fragments without any adapters cannot hybridize to surface bound primers in the flow cell Fragments with an adapter on only one end can hybridize to surface bound primers but cannot form clusters Consumables Item Quantity Storage Supplied By PCR Master Mix PMM 1 tube per 48 25 C to 15 C Ilumina reactions PCR Primer Cocktail PPC 25 C to 15 C Ilumina Resuspension Buffer RSB 1 tube 2 C to 8 C Illumina 96 well HSP plate 1 E e SUE User 96 well MIDI plate 1 15 C to 30 C User AMPure XP beads 50 ul per sample AC O ACE User Freshly prepared 80 ethanol 400 ul per sample 15 C to 30 C User EtOH Ice bucket As needed 2596 to 152 User Microseal A film 1 15 C to 30 C User Microseal B adhesive seals 3 1556 O SAS User Part 15049525 Rev B Item Quantity Storage Supplied By RNase DNase free eight tube 5 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent 5 IBC to SUE User reservoirs if using multichannel pipettes Preparation Prepare an ice bucket Remove the PCR Master Mix and PCR Primer Cocktail from 25 C to 15 C storage Thaw them at room temperature and then place them on ice Centrifuge the thawed PCR Master Mix and PCR Primer C
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12. TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC lumina San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com
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15. incubations starting at 94 C then decreasing 2 C per cycle d 58 C for forever 4 NOTE Run the 58 C hybridization for 90 minutes The RNA HYB program is set to 58 C for forever to make sure that the sample is at 58 C when the plate is removed 6 Immediately remove the plate from the thermal cycler after 90 minutes of 58 C hybridization and proceed immediately to First Capture on page 43 NOTE The total run time of the RNA HYB program is approximately 2 hours Hybridizing longer than 2 hours results in a high degree of non specific binding A 2 Part 15049525 Rev B First Capture This process uses streptavidin beads to capture probes hybridized to the targeted regions of interest Two heated wash procedures remove non specific binding from the beads The enriched library is then eluted from the beads and prepared for a second round of hybridization Consumables Item 2N NaOH HP3 Elute Target Buffer 2 ET2 Enrichment Elution Buffer 1 EE1 Enrichment Wash Solution EWS Streptavidin Magnetic Beads SMB 1 7 ml microcentrifuge tube 96 well HSP plate 96 well MIDI plate Microseal B adhesive seals Preparation Quantity 1 tube 1 tube 1 tube 1 tube 1 tube 1 1 Storage 25 C to 15 C PAC O tC 25 C to 15 C PIC ue SAC 2 C to 8 C IDC to 302 15 C to 30 C TIC ito STRE 15 C to 30 C Supplied By Ilumina Ilumina Ilumina Ilumina Illumina User User User User Remove the 2N NaO
16. indemnity if any to Purchaser Part 15049525 Rev B Revision History Part Revision Date Description of Change 15049525 B July 2014 Removed instructions regarding barcodes and replaced with instructions to label plate with a pen Modified the following plate names e NEH1 changed to RAH1 RNA Access Hyb 1 e NEW1 changed to RAW1 RNA Access Wash 1 NEH2 changed to RAH2 RNA Access Hyb 2 NEW2 changed to RAW2 RNA Access Wash 2 e NECI changed to RACI RNA Access Clean Up 1 NEA changed to RAA RNA Access Amplification NEC2 changed to RAC2 RNA Access Clean Up 2 NEL changed to RAL RNA Access Library Replaced DEPC treated water with nuclease free water to Fragment RNA To Clean Up ALP Changed CAP plate Resuspension Buffer volume from 17 5 ul to 22 5 ul e Changed final supernatant transfer volume from 15 ul to 20 ul Included initial 98 C for 30 seconds setting in Preparation steps for First PCR Amplification Added AMPure XP beads to Consumables and Equipment Removed distilled water and PCR grade water from Consumables and Equipment 15049525 A April 2014 Initial Release TruSeq RNA Access Library Prep Guide VI Part 15049525 Rev B Table of Contents Chapter 1 Overview Introduction caca aaa RNA Input Recommendations suuuueueuusse Additional Resources 0 22 eee eee cee cece ceeccceeeeeseees Chapter 2 Protocol o oaia a
17. process ligates indexing adapters to the ends of the ds cDNA preparing them for hybridization onto a flow cell Consumables Item RNA Adapter Indexes AR001 AR016 AR018 AR023 AR025 AR027 Ligation Mix LIG Resuspension Buffer RSB Stop Ligation Buffer STL 96 well HSP plate 96 well MIDI plate AMPure XP beads Freshly prepared 8076 ethanol EtOH Microseal B adhesive seals RNase DNase free eight tube strips and caps if using multichannel pipettes RNase DNase free reagent reservoirs if using multichannel pipettes Quantity 1 tube of each index being used per column of 8 reactions 1 tube per 48 reactions 1 tube 1 tube per 48 reactions 1 1 92 ul per sample 800 ul per sample 4 28 4 28 Storage 25 C to 15 C 25 C to 15 C 2 C to 8 C 25 C to 15 C 15 C to 30 C ISAC te BOC 2 C to 8 C TIC O XU 15 C to 30 C TIS O EUA 15 C to 30 C Supplied By Ilumina Ilumina Ilumina Ilumina ser ser ser oan Chen C ser E ser User User Part 15049525 Rev B Add LIG Preparation BR ON Remove the following from 25 C to 15 C storage and thaw them at room temperature RNA Adapter tubes depending on the RNA Adapter Indexes being used z NOTE i Review the TruSeq Sample Preparation Pooling Guide part 15042173 See for information on how to download the guide from the Illumina website
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19. ul of First Strand Synthesis Mix Act D and SuperScript II mix to each well of the DFP plate Mix thoroughly as follows a Seal the DEP plate with a Microseal B adhesive seal b Shake the DFP plate on a microplate shaker continuously at 1600 rpm for 20 seconds Return the First Strand Synthesis Mix Act D tube to 25 C to 15 C storage immediately after use Part 15049525 Rev B Incubate 2 DFP 1 Place the sealed DEP plate on the pre programmed thermal cycler Close the lid and select Synthesize 1st Strand a b c d e Choose the pre heat lid option and set to 100 C 25 C for 10 minutes 42 C for 15 minutes 70 C for 15 minutes Hold at 4 C 2 When the thermal cycler reaches 4 C remove the DFP plate from the thermal cycler and proceed immediately to Synthesize Second Strand cDNA on page 20 TruSeq RNA Access Library Prep Guide 1 O VNQ pues 1SJI4 eziseuu S Protocol Synthesize Second Strand cDNA 20 This process removes the RNA template and synthesizes a replacement strand incorporating dUTP in place of dTTP to generate ds cDNA The incorporation of dUTP quenches the second strand during amplification because the polymerase does not incorporate past this nucleotide AMPure XP beads are used to separate the ds cDNA from the second strand reaction mix At the end of this process you have blunt ended cDNA Consumables Item Quantity Storage Supplied By Resuspension Buffer RSB 1 tube 2 C to 8 C Il
20. well of the ALP plate 6 Return the Ligation Mix tube to 25 C to 15 C storage immediately after use 7 Add 2 5 ul thawed RNA Adapter Index to each well of the ALP plate 8 Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes 9 Centrifuge the ALP plate at 280 x g for 1 minute Incubate 2 ALP 1 Place the sealed ALP plate on the pre heated microheating system Close the lid and incubate at 30 C for 10 minutes 2 Remove the ALP plate from the microheating system Add STL 1 Remove the adhesive seal from the ALP plate 2 Add5 ul Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation mix Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes 3 Centrifuge the ALP plate at 280 x g for 1 minute Clean Up ALP 1 Remove the adhesive seal from the ALP plate 2 Vortex the AMPure XP beads for at least 1 minute or until they are well dispersed 3 Add 42 ul mixed AMPure XP beads to each well of the ALP plate Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes 4 Incubate the ALP plate at room temperature for 5 minutes 28 Part 15049525 Rev B 10 11 12 13 14 15 16 17 18 19 20 Centrifuge the A
21. 25 C to 15 C Illumina Resuspension Buffer RSB 1 tube 2 C to 8 C Ilumina Microseal B adhesive seal 1 IBC tro SUE User Preparation Remove the following from 25 C to 15 C storage and thaw them at room temperature Capture Target Buffer 3 Coding Exome Oligos Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature 1 Remove the RAH2 plate from 25 C to 15 C storage if it was stored at the conclusion of First Capture and thaw on ice Centrifuge the thawed RAH2 plate at 280 x g for 1 minute Thoroughly vortex the Capture Target Buffer 3 tube until the solution is completely resuspended Visually make sure that no crystal structures are present NOTE f If crystals and cloudiness are observed vortex the Capture Target Buffer 3 tube until it is clear Remove the adhesive seal from the RAH2 plate Part 15049525 Rev B Add the following reagents in the order listed to each well of the RAH2 plate Reagent Volume ul Resuspension Buffer 20 Capture Target Buffer 3 50 Coding Exome Oligos 5 Mix thoroughly as follows a Seal the RAH2 plate with a Microseal B adhesive seal Make sure that the plate is tightly sealed to prevent potential evaporation Use an adhesive seal roller to apply force to the seal and make sure that the seal is secured b Shake the RAH2 plate on a microplate shaker at 1200 rpm for 1 minute Centrifuge the RAH2 plate at 280 x g for 1 minute Place the seale
22. 6 Part 15049525 Rev B Second PCR Amplification This process uses PCR to amplify the enriched DNA library for sequencing Consumables Item Quantity Storage Supplied By Enhanced PCR Mix EPM 1 tube 25 C to 15 C Ilumina PCR Primer Cocktail PPC 1 tube 25 C to 15 C Ilumina Microseal A film 1 15 C to 30 C User Microseal B adhesive seal 1 TIC to ABE User Preparation Remove the Enhanced PCR Mix and PCR Primer Cocktail from 25 C to 15 C storage and thaw on ice Briefly centrifuge the thawed Enhanced PCR Mix and PCR Primer Cocktail tubes for 5 seconds 4 NOTE If you do not intend to consume the Enhanced PCR Mix and PCR Primer Cocktail in one use dispense the reagents into single use aliquots Freeze the aliquots to avoid repeated freeze thaw cycles Remove the RAA plate from 25 C to 15 C storage if it was stored at the conclusion of Second Capture and thaw on ice Centrifuge the thawed RAA plate at 280 x g for 1 minute Remove the adhesive seal from the thawed RAA plate TruSeq RNA Access Library Prep Guide 5 T uoneodwy yO d PUODES Pre program the thermal cycler with the following program and save as the EPM AMP program Choose the pre heat lid option and set to 100 C 98 C for 30 seconds 10 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 10 C y NOTE Illumina has optimized the number of recommended PCR cycles for enrichment assays based on the level o
23. A EU TTAAGAGCTACCGTGCAACGACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCT TA JAUNES A LA CGTTAAGATTACTTGATCCACTGATTCAACG TACCGTAACGAACG TATCAAT TGAGCTICTGTIAACCTTY ASALTAT TE EAT TAL Agia AAC RON TCAATTGAGACTAGCAACGACQG NIN AAA CATA CACACICTGTIRAC TE o Near GTAACGAACGTATCAAT TGAGACTAAATATTAACGTACCAT TAAGAGCTACC SATAA AQTRACACAC TTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT T GAGACTAAA AN KAGAGC TACCGTCTTCTGTIAACCTIAAGATIACTTGA YER SATI a STACCGTAACGAACGIA TCAT TAAGAT TACTTGATCCACT GATT CAACGTACCGTAACGAACG TAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACTTCTGTTAACCTT IAAAAGAATGATAACAGTAACACACT TCTGTTAACC T TAAGAT TACT TGATCCACTGATT CAACGTACCGTAAAGAT TACT Ti GATCCACTGAT I GAACCTACCGTAACGAACGTAT CAME SARAC AA TA TAACGTACGAT TMACAGCTACO CCAT TAAGAGCTACCGTGCAACAGTAACACACT TCTGTTAACCTTAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAA 3ATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGATCCACTGATTCAACGTACCG TAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACG Z2LTGATCCACTGATTCAACGTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGT TAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAGCAACGACQ ERAT TARTE ACT ET T TA GA TOTI GATE GECI GATIG a O DARET C ACA AA ON Galt ore aL GTACCGTAACGAACGTATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGA P pe E ACCGTGCAACGACGAAAAGAAT
24. ACTGATTCAACG TACCG TAACGAACG TATCAAT T GAGAC TAAATAT TAACG TACCAT TAAGAGCTA TTCTG CCTTAAGATTACT TGATCCACTGA CCAT TAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAACTTCTGT TAACCT TAAGAT TACTTGAT CTAGOGIGCAAGGAAAN AAGC TTAAGATTACT IGATCCACTGAT ICAACGTAGTICTG TAAGGTTAAGATTACT IGATCCACT GAT CAACGACOGTAACGAACG TAI GAAT TGAGAG TAAGGAGCGIGCAACGACGAAAAGAATAT AAAAGAATGA NOAA Ca IPA UL AACCTIAAGATTACTT EE AE GMT S M AAAGATTACTTGAT VER ACTGATICAACGTACCGTAACGAACG TA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACTTCTGTTAACCT TAAGAT TACT T PORT OCACTGALICAACGT ACCGTAACGAA ACTA TCAAT TGAGACTAAATAT TAACGT AC CATAAGAGCTACCGTOCAA AOGA OINA GA TAACAGTAACACACTTCTGTI CCATTAAGAGCTACEGT SC AACAGTAACACAC E TGTOT IAACCTTAAGATTACT GAT GGAGTGATTCAA OTACOG IAACGAACG IAT CAAT GAGAGTAAATAT TAACGTACGAI TAAGAGCTACCG I GCAACGACGARAAGAAT SATAN 3ATAACAGTAACACACT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT T GAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCT AAGAT T BOT GAT RG SATI ONG aTACCGT AA ANC ATCATTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC TAAATAT TAACG TACCATTAAGAGC TACCGTGCAACGA CORABACAA AGAT TAACAGTAACACACTTCTGTTAACCT T STTGATOCAGT GAIT GAACG TTAAGATTACTTGATCCACTGATT GAACGTACCGTAACGAACGTATCAATT GAGCTTCTG HACC AAGAT TACT IGATCCAG TGA CAAGOTACCGTAACGAACG TA CARTTGAGACTAGGAACGACG IAAAAGAATGATAACAGTAACACACT TCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGAT
25. AT TCAACGTACCGTAACGAAC ACGAAAAGAATGATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATATTAACGTACCAT TAAGAGCTACCGT AATGATAACAG TAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATATTAACGTA AE al Soa ea iu AAGATTACTTGATCCACT GAT TCAACGTA AAT TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATATTAACGTACCATTAAGAGCT TCTGT TAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAC CGTATGAA GAGACTAMATA AAC TACTTAAOS TARGATTACT I GATCCAGTGATT CAACG IACCGIAACGAACG ICT TCTGT TAAGC JAAGATTACT IGATCCACTGAT TOMOGTACOSTACGAACO TATGAAT GAGACTAACGACGANS GAGACTAAATAT TAACGTACCAT TAAGAGCTACAACCTTAAGA BEC SS OSEE S COP pne TCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGCTACCGTGCAACGACGAAAAGAAT GA E AATGATAACAGTAACACACT TCTGTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCG TIAACGAACG TA AT GAGAG MATA TAAG QUAC CAT DAC GC TAGCG GTC TT ACT AGAT TAG SATA AACG GTACCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT AI TA AC PACALA Aaaa RIC AIA e I EE al AACCTT EXCEL AME I EU M CTGTTAACCTTAAGATTACTTGATCCAC GAAAAGAATGATAA ACGAAAAGAATGATAACAG TAACACAC TTCTGT TAACCT T TTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGAT C II TAAGAGCTACCGT AATGATAACAGTAACACACT TCTGT TAACCTTAAG ACA C CACTGA T CAACGTACCOTAACGAACG TAT CANT GAGAG TA CACACTTCTGTTAA Arp e a e ne E y AATGATAACA AATGATAACAGTAACACACTTCTGTTAACCTT TTGAT
26. CCACTGATTCAACGTACCGTAACGAA TCAATTGAGACTA T AGEA AA RAE TACA TARA TTACTIONTOCACICATICAACGTA CO IRACCAACGIATCARTTGAGACTAAATATH A ITACT TGATCCACTGATTCAACGT TAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAAt CGAA ACGAAAAGAATGATAACAGTAACACACT TCTGT TAACCTTAAGAT TACTT GATCCACTGATT CAACGTACCGTAAAGAT TACTTGATC AAGAGCTACCGT FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY RS 301 9001DOC Part 15049525 Rev B July 2014 This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT
27. Capture ae acea 2 aci ceases aa a a aaa da aa aaa d it i aa 23 a a aaa a 50 Capture Sample Clean Up aaa 54 Second PCR Amplification aaa aaa 57 Second PCR Clean Up aaa 60 Validate Library uuuuuuuuuuuuussseeslesslsIIII aaa 63 a AA T E amen E EET 3 A E K F AT ome o Sen PE gt NM YW 2x AA A I eger 007 gt AA A fi OST i ses TR E Zi LLL e a rfi A F A TGCGGC rA rogag c6 TAa acon e a E dan d pre aor ceca s E cocoa j aT S y T TruSeqRNA Access Library Prep Guide 9 c Je1deuo Protocol Introduction 10 This chapter describes the TruSeq RNA Access Library Prep protocol Review Best Practices before proceeding See Additional Resources on page 7 for information on how to access TruSeq RNA Access Library Prep Best Practices on the Illumina website Follow the protocols in the order shown using the specified volumes and incubation parameters If you are pooling record information about your samples before beginning library prep for later use in data analysis Use IEM to create and edit sample sheets for Illumina sequencing systems and analysis software See Additional Resources on page 7 for information on how to download IEM software and documentation from the Illumina website TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 7 for information on how to download the guide from the Illumina website Review Appendix A Support
28. GAT TCAACG TACCG TAACGAACG TATCAAT TGAGA CTAAATALTAACGTACCAT AAGAGCTACUGTCITCTGI TANGGA IAAGATIACT TGATSCACTGA GAACGTA CO VAS CL LM poe ime M E ACCGTAACGAACQG TATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGC TACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TCTGTTAACCTT ACGAAAAGAATGATAACAG TAACACACT TCTGT TAACCT TAAGAT TACTT GAT CCACTGATTCAACGTACCGTAAAGAT TACT TGATCCACTGAT I CAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACG RAZA AAA AAA ATACAT IO AGE IA al MAG ABEL ETT T TA TASTIERA TRA T AT E TRA ACETAT MAT SAGA TAATAI CE IN GAT IMAGE PAGE TGA e GOGA AGA GN TAA AATGATAACAGTAACACACT TCTGT TAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCG TAACGAACGTAT CAAT TGAGAC TAAATAT TAACG TACCAT TAAGAGC TACCG TCTTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTA ITACTTGATCCACTGAT TCAACGTTIAAGAT TACTT GAT CCACT GATT CAACGTACCGTAACGAACGTATCAATT GAGCT TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT I GAGACTAGCAACGACGAA ACC C G AACGACGAAAAGAATGAT ACACTTCTGTTAACC MIACTTOMCCACTEATTCAACGTTAAGALTACTTGATCCACT GATTCAAGGTACCGTAACGAACGTATCAATIGAGCHICTOLL AACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAGCAACGACGAA AAA O RCM TRAD IEC CAI Ac ARAS TAI GAT EA TAAA TRAC Ue CAT TA ACEL TACI AATGATAACAGTAACACACT TC TGTTAACCT TAAGAT TA SABCYN A A eU EL e CTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAA TCCACTGAT T CAACGTACCAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGTCTTCTGT TAACCT TAAGAT TACI TGATCCACT G
29. GATAACAG TAACACACT TCTGT TAACCT A ZTTGATCCACTGATTCAACGTTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGCTTCTGT TAACCT TAAGAT TACT TGAT CCACTGAT TCAACG TACCGTAACGAACGTAT CAAT TGAGACTAGCAACGACG IAAAAGAATGATAACAGTAACACAC T TCTGT TAACCT TAAGAT TACTT GATCCACTGAT TCAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC 3ATAACAGTAACACACT TCTGTTAACCT TAAGAT TA quU A di eie SIL RE I Real AR SCIRE MAUS ARES ATTACTT OE Oe cU E ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCG TAACGA AAAAGANTGATAACAGTAAGAGAGTIGTGY IAACCLTAAGATIACT TGATCGACT GATT CA ACQTAOGE TRAGA AC EAI CEAC GAT ICAAGGTACCGTAACGAACGIATCAAI TGAGAC TAAATATTAACGTAGCATTAAGAGC AGO Peer ess use DA DRE REZ GTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAA a ENA Aga A SEU ene APTE ARR eiue GATTACTTGATCCACTGATTCAACG TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATA LU ae aa GCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCG TA ATCAATTGAGA CTAANTALT AACGTACTTAACCTIAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TCTTCTGT TAACCT TAAGAT T KETIGMCCACIGATTCAACGT ACCGTAACGAACGTATCAAT TGAGACTAACGACGA SATAACAGTAACAGAG PRET ARCOT TAAGA TACT GAT GOAGTGAT GAACGIAGGG TARO GAO O TALCA GAGAG AAATAT TAA DG ACCA TAAGAGCTACGGCT TCT GTTAAGG TANGATIACT IGATCUACT GAT IGAACG 3ATAACAGTAACACACTT AACCTTAAGATTACTTGATCC
30. H Enrichment Elution Buffer 1 and Enrichment Wash Solution from 25 C to 15 C storage and thaw at room temperature Remove the Elute Target Buffer 2 and Streptavidin Magnetic Beads from 2 C to 8 C storage and let stand at room temperature Pre heat the microheating system to 50 C TruSeq RNA Access Library Prep Guide 43 31n1de 18 114 Protocol First Bind 10 11 12 44 Label a new 96 well MIDI plate RAW1 RNA Access Wash 1 with a smudge resistant pen Label a new 96 well HSP plate RAH2 RNA Access Hyb 2 with a smudge resistant pen Remove the RAHI plate from the thermal cycler Centrifuge the RAHI plate at 280 x g for 1 minute Remove the adhesive seal from the RAH1 plate Take care when removing the seal to avoid spilling the contents of the wells Transfer the entire contents 100 ul from each well of the RAH1 plate to the corresponding well of the new 96 well MIDI plate labeled RAW1 Vortex the Streptavidin Magnetic Beads tube until the beads are well dispersed then add 250 ul well mixed Streptavidin Magnetic Beads to the wells of the RAW1 plate Mix thoroughly as follows a Seal the RAW1 plate with a Microseal B adhesive seal b Shake the RAW1 plate on a microplate shaker at 1200 rpm for 5 minutes Let the RAW1 plate stand at room temperature for 25 minutes Centrifuge the RAW1 plate at 280 x g for 1 minute Remove the adhesive seal from the RAW plate Place the RAW1 plate on the magnetic st
31. LIGENCE STRICT LIABILITY OR OTHERWISE ILLUMINA S TOTAL AND CUMULATIVE LIABILITY TO PURCHASER OR ANY THIRD PARTY ARISING OUT OF OR IN CONNECTION WITH THESE TERMS AND CONDITIONS INCLUDING WITHOUT LIMITATION THIS PRODUCT INCLUDING USE THEREOF AND ILLUMINA S PERFORMANCE HEREUNDER WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE SHALL IN NO EVENT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT Limitations on Illumina Provided Warranties TO THE EXTENT PERMITTED BY LAW AND SUBJECT TO THE EXPRESS PRODUCT WARRANTY MADE HEREIN ILLUMINA MAKES NO AND EXPRESSLY DISCLAIMS ALL WARRANTIES EXPRESS IMPLIED OR STATUTORY WITH RESPECT TO THIS PRODUCT INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NONINFRINGEMENT OR ARISING FROM COURSE OF PERFORMANCE DEALING USAGE OR TRADE WITHOUT LIMITING THE GENERALITY OF THE FOREGOING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES Product Warranty All warranties are personal to the Purchaser and may not be transferred or assigned to a third party including an affiliate of Purchaser All warranties are facility specific and do not transfer if the Product is moved to another facility of Purchaser unless Illumina conducts such move a Warranty for Consumables Illumina warrants that Consumables other than custom Consumables will conform to
32. LP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 79 5 ul of supernatant from each well of the ALP plate Take care not to disturb the beads NOTE Leave the ALP plate on the magnetic stand while performing the following 8076 EtOH wash steps 9 11 With the ALP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads sJo1depy e1e6r Incubate the ALP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 9 and 10 one time for a total of two 8076 EtOH washes With the ALP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes Remove the ALP plate from the magnetic stand Add 52 5 ul Resuspension Buffer to each well of the ALP plate Mix thoroughly as follows a Seal the ALP plate with a Microseal B adhesive seal b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the ALP plate at room temperature for 2 minutes Centrifuge the ALP plate at 280 x g for 1 minute Remove the adhesive seal from the ALP plate Place the ALP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Transfer 50 ul of supernatant from each well of t
33. RAW2 plate stand at room temperature for 25 minutes 8 Centrifuge the RAW2 plate at 280 x g for 1 minute 9 Remove the adhesive seal from the RAW2 plate 10 Place the RAW2 plate on the magnetic stand for 2 minutes at room temperature or until the liquid is clear 11 Carefully remove and discard all of the supernatant from each well of the RAW2 plate without disturbing the beads 12 Remove the RAW2 plate from the magnetic stand Second Wash 1 Make sure that the Enrichment Wash Solution tube is at room temperature then thoroughly vortex the tube NOTE It is normal that the Enrichment Wash Solution can be cloudy after vortexing 2 Add 200 ul Enrichment Wash Solution to each well of the RAW2 plate TruSeq RNA Access Library Prep Guide 5 1 eJnjde puogas Protocol Mix thoroughly as follows a Seal the RAW2 plate with a Microseal B adhesive seal b Shake the RAW2 plate on a microplate shaker at 1800 rpm for 4 minutes c Remove the adhesive seal from the RAW2 plate d Gently pipette the entire volume of each well up and down to ensure complete resuspension of the sample Seal the RAW2 plate with a Microseal B adhesive seal Incubate the RAW2 plate on the pre heated microheating system with the lid closed at 50 C for 20 minutes Place the magnetic stand next to the microheating system for immediate access Remove the RAW2 plate from the microheating system and immediately place it on the magnetic stand for 2 minu
34. TA AA GA ae TOT CETE TRAG TI ee oe UNG a Leg PARIS EL GTACCATTAAGAGC TACCGTGCAACTTAACCTTAAGAT TACTTGATCCACT GAT TCAACGTACCGTAACGAACGTATCAATT GAGACTAAATAT TAACGTACCA WA AU as CGACG TICTGTT POM Seu GAGCTACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAACGAACGTA TRATIGAGACTAAGCIACCOTECAACGA CGAAAAGAATGAT A GRAAAGAATQATARCAGTARCACAG CTO IAACCTTAAGATTACT TGATCCACTGAT TORACOTACOS MAGA TIACITGATOCAS FSA GAASG ACG TAACGAAUGTAT CATT GAGACTAAATATTAACGIAGCAT TAA GAG TACCA AATGATAACAGTAACACACT TCTGTTAACCTTAAGA uod Les CCGTAACGAACG TATCAAT TGAGACT i OVAL pe re ve CIE CGACGAAAAGAA Ap para Ura preg SEN GTACCAT TAAGAGC TACCGTGCAACAGTAACACACT TCTGT TAACCT TAAGAT TACTT GATCCACT GAT TCAACGTACCGTAACGAACGTAT CAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGT GCAACGACGAAAAGAAT GATAACA AAT OATAACAGSTAACAGACTICNG TIAAGGTIAAGATTAC LEGATCCAGT CAT CANCG TACCO TAACGAACGTAT GANTT GAGA AAA IA ACT AAA LANGA CE SLOT MAACO TIAAGATIACH GATCCACTGATTCARCTA ACGTACCGTAACGAACGTATCAT TAAGAT TA ea ce Co m pe T ni CLP NECS E CGAAAAGAATGA a CTTCTGTTAACCTTAAC I TACTTGATCCACTGATTCAACGTTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG AT CAAT TGAGCTTCTGTTAACCT TAAGAT TACT TGATCCACT EM EA I SES VA TCAATTGAGACTAGCAACGACGAA ACGAAAAGAATGATAACAGTAACACAC T TCTGTTAACCT TAAGAT TACTT GATCCACTGATT CAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACG TACCGTAACGAACG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGC TACCGT AATGATAACAG TAACACACT TC TGT TAACCT TAAGAT TACT TGATCCACT
35. a recommends using a 20 ul single channel or multichannel pipette set to 10 5 ul to perform two consecutive transfers of 10 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads 9 Add 4 ul Elute Target Buffer 2 to each well of the RACI plate containing samples to neutralize the elution 10 Mix thoroughly as follows a Seal the RACI plate with a Microseal B adhesive seal b Shake the RACI plate on a microplate shaker at 1800 rpm for 1 minute 11 Centrifuge the RACI plate at 280 x g for 1 minute 12 Store the remaining reagents as follows a Place the Elute Target Buffer 2 and Streptavidin Magnetic Beads tubes in 2 C to 8 C storage b Place the 2N NaOH Enrichment Elution Buffer 1 and Enrichment Wash Solution tubes in 25 C to 15 C storage c Discard any remaining elution pre mix TruSeq RNA Access Library Prep Guide 5 3 eJnjde puogas Q Capture Sample Clean Up O This process uses AMPure XP beads to purify the captured library before PCR amplification Consumables Item Quantity Storage Supplied By Resuspension Buffer RSB 1 tube 2 C to 8 C Ilumina 96 well HSP plate 1 TIC tie SOE User AMPure XP beads 45 ul per sample 2 C to 8 C User Freshly prepared 80 ethanol 400 ul per sample 15 to 30 C User EtOH Microseal B adhesive seals 3 15 C to 30 C User Preparation Procedure o4 Remove the Resuspension Buffer and AMPure XP beads fro
36. age of RNA fragments gt 200 nt DV as a reliable determinant of FFPE RNA quality Y WARNING If starting with FFPE RNA do not perform the Incubate 1 DFP steps in this procedure Table 2 FFPE RNA Input Recommendations Quality DV Input Requirement Per Reaction High gt 70 20 ng Medium 50 70 20 40 ng Low 30 50 40 100 ng Too Degraded lt 30 Not recommended dy NOTE For more information see the Evaluating RNA Quality from FFPE Samples tech note for TruSeq RNA Access Library Prep See Additional Resources on page 7 for information on how to download the tech note from the Illumina website TruSeq RNA Access Library Prep Guide 1 3 YNY jJuaubely Protocol Consumables Item Elute Prime Fragment High Mix EPH Resuspension Buffer RSB 96 well HSP plate Microseal B adhesive seal Total RNA RNase DNase free eight tube strips and caps if using multichannel pipettes RNase DNase free reagent reservoirs if using multichannel pipettes Nuclease free ultra pure water Quantity 1 tube per 48 reactions 1 tube 1 jl 10 ng fresh frozen RNA per reaction Or 20 100 ng FFPE RNA per reaction see Table 2 1 Enough to dilute each total RNA sample to a final volume of 8 5 ul Storage 25 C to 15 C 25 C to 15 C 15 C to 30 C TIC 0 HOC 15 C to 30 C ISS te SAC 15 C to 30 C FE tose Supplied By Illumina Illumina User User User User User User P
37. allow for pooling up to 24 samples using the 12 different indexes in each kit i NOTE The TruSeq RNA Access contains enough of each reagent for 4 plex pooling Pooling at less than 4 plex results in the inability to process the number of samples supported by the kit Table 5 TruSeq RNA Access Kits Kit Name TruSeq RNA Access Library Prep Kit Set A TruSeq RNA Access Library Prep Kit Set B TruSeq RNA Access Library Prep Kit Catalog Number Number of of Samples Indexes Supported RS 301 2001 48 12 RS 301 2002 48 12 The TruSeq RNA Access Library Prep Kit contains four boxes cDNA Synthesis PCR Box 1 12 Index Set A or B Box 2 Capture Reagents Box 3 Coding Transcriptome Box 4 7O Part 15049525 Rev B cDNA Synthesis PCR Box 1 Store at 25 C to 15 C This box is shipped on dry ice As soon as you receive your kit store the components at 25 C to 15 C Figure 10 TruSeq RNA Access cDNA Synthesis PCR 48 Samples Box 1 part 15052309 Lo 15026785 PCR Master Mix 15031748 PCR Primer Cocktail 15031094 First Strand Synthesis Act D Mix 15031098 Second Strand Marking Master Mix TruSeq RNA Access Library Prep Guide 11 S1U91U02 1M Supporting Information 72 12 Index Set Box 2 You receive either a Set A or Set B Box 2 in the kit depending on the set ordered Store at 25 C to 15 C These boxes are shipped on dry ice As soon as you receive your kit store
38. an Up Consumables AMPure XP Beads RSB Plates RAC2 RAL a 1 Validate Library 11 MO PILJOM deJg Mea Protocol 12 Prepare Adapter Setup Use IEM or BaseSpace to record information about your samples before beginning library preparation Do one of the following Use IEM to create and edit sample sheets for Illumina sequencing systems and analysis software See Additional Resources on page 7 for information on how to download IEM software and documentation from the Illumina website Use BaseSpace to organize samples libraries pools and a run for Illumina sequencing systems and analysis software See Additional Resources on page 7 for information on how to access BaseSpace or download BaseSpace documentation from the Illumina website Review planning steps in the TruSeq Sample Preparation Pooling Guide part 15042173 See Additional Resources on page 7 for information on how to download the guide from the Illumina website Illumina recommends arranging samples that will be combined into a common pool in the same row Include a common index in each column This arrangement facilitates pipetting operations when dispensing indexed adapters and pooling indexed libraries later in the protocol Part 15049525 Rev B Fragment HNA This process fragments and primes RNA for cDNA synthesis i NOTE If starting with FFPE RNA the sample input amount is based on sample quality Illumina recommends using the percent
39. and for 2 minutes at room temperature or until the liquid is clear Carefully remove and discard all of the supernatant from each well of the RAW1 plate without disturbing the beads Remove the RAW1 plate from the magnetic stand Part 15049525 Rev B First Wash 1 Make sure that the Enrichment Wash Solution tube is at room temperature then thoroughly vortex the tube E NOTE i It is normal that the Enrichment Wash Solution can be cloudy after vortexing 2 Add 200 ul Enrichment Wash Solution to each well of the RAW1 plate 391n1de 18 114 3 Mix thoroughly as follows a Seal the RAW1 plate with a Microseal B adhesive seal b Shake the RAWI plate on a microplate shaker at 1800 rpm for 4 minutes c Remove the adhesive seal from the RAW plate d Gently pipette the entire volume of each well up and down to ensure complete resuspension of the sample 4 Seal the RAW1 plate with a Microseal B adhesive seal 5 Place the sealed RAWI plate on the pre heated microheating system Close the lid and incubate at 50 C for 20 minutes 6 Place the magnetic stand next to the microheating system for immediate access 7 Remove the RAW1 plate from the microheating system and immediately place it on the magnetic stand for 2 minutes or until the liquid is clear 8 Remove the adhesive seal from the RAW1 plate 9 Immediately remove and discard all of the supernatant from each well of the RAW1 plate 10 Remove the RAW1 plate from th
40. art 15049525 Rev B Preparation Make DFP 1 Remove the following from 25 C to 15 C storage and thaw them at room temperature Elute Prime Fragment High Mix Resuspension Buffer 4 NOTE The Resuspension Buffer can be stored at 2 C to 8 C after the initial thaw Pre program the thermal cycler with the following program and save as Elution 2 Frag Prime Choose the pre heat lid option and set to 100 C 94 C for 8 minutes 4 C hold Set the centrifuge to 15 C to 25 C if refrigerated Label a new 96 well HSP plate DFP Depleted RNA Fragmentation Plate with a smudge resistant pen Dilute total RNA with nuclease free ultra pure water to a final volume of 8 5 ul in each well of the new 96 well HSP plate labeled DFP Add 8 5 ul Elute Prime Fragment High Mix to each well of the DFP plate Mix thoroughly as follows a Seal the DFP plate with a Microseal B adhesive seal b Shake the DFP plate on a microplate shaker continuously at 1600 rpm for 20 seconds Return the Elute Prime Fragment High Mix to 25 C to 15 C storage TruSeq RNA Access Library Prep Guide 1 D YNY tusuu Ge Protocol Incubate 1 DFP Y WARNING If starting with FFPE RNA do not perform this incubation procedure Proceed immediately to Synthesize First Strand cDNA on page 17 1 Place the sealed DEP plate on the pre programmed thermal cycler Close the lid and select Elution 2 Frag Prime to fragment and prime the RNA a Choose th
41. authorized resellers and distributors conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any claims in the foregoing patents or patent applications directed to producing the product The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES 1 use of the product or its components in manufacturing or 2 use of the product or its components for therapeutic or prophylactic purposes in humans or animals Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Illumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Illumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Illumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all possible fields of use the Core IP App
42. d RAH2 plate on the pre programmed thermal cycler Close the lid then select and run the RNA HYB program a Choose the pre heat lid option and set to 100 C b 95 C for 10 minutes c 18 cycles of 1 minute incubations starting at 94 C then decreasing 2 C per cycle d 58 C for forever x NOTE Run the 58 C hybridization for 90 minutes The RNA HYB program is set to 58 C for forever to make sure that the sample is at 58 C when the plate is removed Immediately remove the plate from the thermal cycler after 90 minutes of 58 C hybridization and proceed immediately to Second Capture on page 50 J NOTE The total run time of the RNA HYB program is approximately 2 hours Hybridizing longer than 2 hours results in a high degree of non specific binding TruSeq RNA Access Library Prep Guide 4 Q UOIJEZIPIUQ H Puodss Protocol Second Capture o0 This process uses streptavidin beads to capture probes hybridized to the targeted regions of interest Two heated wash procedures remove non specific binding from the beads The enriched library is then eluted from the beads and prepared for sequencing 1 NOTE These procedures are similar to the First Capture on page 43 Consumables Item Quantity Storage Supplied By 2N NaOH HP3 1 tube 25 C to 15 C Illumina Elute Target Buffer 2 ET2 1 tube 2AC o EE Ilumina Enrichment Elution Buffer 1 EE1 1 tube 25 C to 15 C Ilumina Enrichment Wash Solution EWS 1 tube 25 C to 15 C Illumina Streptavidi
43. diately to Validate Library on page 37 you can safely stop the protocol here If you are stopping seal the TSP1 plate with a Microseal B adhesive seal and store at 25 C to 15 C for up to 7 days 3 6 Part 15049525 Rev B Validate Library Illumina recommends performing the following procedures for quality control analysis on your sample library and quantification of the DNA library templates Quantify Library Quantify your library using an Advanced Analytical Fragment Analyzer or Agilent Technologies 2100 Bioanalyzer As an alternative quantify using PicoGreen Quality Control 1 Do one of the following Dilute 1 ul of resuspended construct with 1 ul Resuspension Buffer and load on an Advanced Analytical Fragment Analyzer using Standard Sensitivity NGS Fragment Analysis Kit Load 1 ul of resuspended construct on an Agilent Technologies 2100 Bioanalyzer using a DNA specific chip such as the Agilent DNA 1000 2 Check the size and purity of the sample The final product should be a band at approximately 260 bp Figure 7 Example of TruSeq RNA Access Library Prep Library Size Distribution 100 ng UHR TruSeq RNA Access Library Prep Guide 3 e Heq e3epi eA Protocol 38 Figure 8 TruSeq RNA Access Library Prep 260 bp PCR Product g E 3 g E 8 Part 15049525 Rev B First Hybridization This process mixes the DNA library with capture probes to targeted regions of interest The recomme
44. e flow cell Optimizing cluster densities requires accurate quantitation of DNA library templates Quantify your libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide part 11322363 NOTE i See Additional Resources on page 7 for information on how to download the Illumina Sequencing Library qPCR Quantification Guide part 11322363 from the Illumina website When quantitation is complete proceed to cluster generation For more information see the cluster generation section of the user guide for your Illumina instrument Optional Assess Quality Do the following to assess library quality 1 Load 1 ul of the post enriched library on one of the following Advanced Analytical Technologies Standard Sensitivity NGS Fragment Analysis Kit Agilent High Sensitivity DNA Chip 2 Check the size of the library for a distribution of DNA fragments with a size range from approximately 200 bp 1 kb Follow manufacturer instructions for either the Advanced Analytical Technologies Fragment Analyzer or Agilent Technologies 2100 Bioanalyzer depending on the kit you are using Depending on the level of indexing insert size distribution can vary slightly however the sample peak must not be significantly shifted compared to the example in Figure 9 TruSeq RNA Access Library Prep Guide 6 3 Heq e3epi eA Protocol 64 Figure 9 Example TruSeq RNA Access Library Prep Post Enrichment Library Distribution
45. e magnetic stand 11 Repeat steps 2 10 one time for a total of two Enrichment Wash Solution washes TruSeq RNA Access Library Prep Guide A 5 Protocol First Elution 46 1 Add the following reagents in the order listed in a new 1 7 ml microcentrifuge tube to create the elution pre mix Multiply each volume by the number of pooled samples being prepared The volumes include an excess amount for processing multiple samples Reagent Volume ul Enrichment Elution Buffer 1 28 5 2N NaOH 1 5 Total volume per enrichment 30 Vortex the elution pre mix tube then add 23 ul of the mix to each well of the RAW1 plate Mix thoroughly as follows a Seal the RAWI plate with a Microseal B adhesive seal b Shake the RAW1 plate on a microplate shaker at 1800 rpm for 2 minutes Let the RAW1 plate stand at room temperature for 2 minutes Centrifuge the RAW1 plate at 280 x g for 1 minute Carefully remove the adhesive seal from the RAW1 plate to avoid spilling the contents of the wells Place the RAW1 plate on the magnetic stand for 2 minutes or until the liquid is clear Transfer 21 ul of clear supernatant from each well of the RAW1 plate to the corresponding well of the new HSP plate labeled RAH2 Take care not to disturb the beads NOTE i Ilumina recommends using a 20 ul single channel or multichannel pipette set to 10 5 ul to perform two consecutive transfers of 10 5 ul This technique reduces sample loss by making sure that al
46. e pre heat lid option and set to 100 C b 94 C for 8 minutes c Hold at 4 C 2 Remove the DFP plate from the thermal cycler when it reaches 4 C and centrifuge briefly 3 Proceed immediately to Synthesize First Strand cDNA on page 17 1 6 Part 15049525 Rev B Synthesize First Strand cDNA This process reverse transcribes the cleaved RNA fragments that were primed with random hexamers into first strand cDNA using reverse transcriptase The addition of actinomycin D to the First Stand Synthesis Act D mix FSA prevents spurious DNA dependent synthesis while allowing RNA dependent synthesis improving strand specificity Consumables Item Quantity Storage Supplied By First Strand Synthesis Act D 1 tube 25 C to 15 C Illumina Mix FSA Microseal B adhesive seal 1 ISAC tt SOC User RNase DNase free eight tube 1 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent il IBC O HOKE User reservoirs if using multichannel pipettes SuperScript II Reverse 1 tube 25 C to 15 C User Transcriptase Y y WARNING First Strand Synthesis Act D Mix contains actinomycin D a toxin Personal injury can occur through inhalation ingestion skin contact and eye contact Dispose of containers and any unused contents in accordance with the governmental safety standards for your region Refer to the safety data sheet SDS for detailed environmental health and safety information SDSs are available on the
47. eed immediately to Adenylate 3 Ends on page 24 you can safely stop the protocol here If you are stopping seal the ALP plate with a Microseal B adhesive seal and store at 25 C to 15 C for up to 7 days TruSeq RNA Access Library Prep Guide 2 3 YNQ2 pueJis puooeg aziseyju s Protocol Adenylate 3 Ends A single A nucleotide is added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction A corresponding single T nucleotide on the 3 end of the adapter provides a complementary overhang for ligating the adapter to the fragment This strategy ensures a low rate of chimera concatenated template formation Consumables Item Quantity Storage A Tailing Mix ATL 1 tube per 48 25 C to 15 C reactions Resuspension Buffer RSB 1 tube DEOS Ice bucket As needed 25 C to 15 C Microseal B adhesive seal il AC to SAS RNase DNase free eight tube 3 15 C to 30 C strips and caps if using multichannel pipettes RNase DNase free reagent B E 10 SAC reservoirs if using multichannel pipettes Preparation Prepare an ice bucket Supplied By Illumina Illumina User User User User Remove the A Tailing Mix from 25 C to 15 C storage Thaw it at room temperature and then place it on ice Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature 24 Part 15049525 Rev B Add ATL
48. eparately installed on or embedded in a Product is licensed to Purchaser and not sold Except as expressly stated in this Section no right or license under any of Illumina s intellectual property rights is or are granted expressly by implication or by estoppel Purchaser is solely responsible for determining whether Purchaser has all intellectual property rights that are necessary for Purchaser s intended uses of this Product including without limitation any rights from third parties or rights to Application Specific IP Illumina makes no guarantee or warranty that purchaser s specific intended uses will not infringe the intellectual property rights of a third party or Application Specific IP 3 Regulatory This Product has not been approved cleared or licensed by the United States Food and Drug Administration or any other regulatory entity whether foreign or domestic for any specific intended use whether research commercial diagnostic or otherwise This Product is labeled For Research Use Only Purchaser must ensure it has any regulatory approvals that are necessary for Purchaser s intended uses of this Product 4 Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Illumina consumables reagents with Illumina Hardware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchaser agrees not to nor authorize any third party to engage in
49. es Item Quantity Storage Supplied By Resuspension Buffer RSB 1 tube 2 C to 8 C Ilumina 96 well HSP plate il IBC 10 SAC User 96 well MIDI plate 1 15 C to 30 C User AMPure XP beads 90 ul per sample AC O BAC User Freshly prepared 80 ethanol 400 ul per sample 15 C to 30 C User EtOH Microseal B adhesive seals 3 TIC ie SU User Preparation Review Best Practices for Handling Magnetic Beads See for information on how to access Best Practices on the Illumina website Remove the Resuspension Buffer and AMPure XP beads from 2 C to 8 C storage and bring them to room temperature Remove the RAA plate from 2 C to 8 C storage if it was stored at the conclusion of Second PCR Amplification and let stand to bring to room temperature Label a new 96 well MIDI plate RAC2 RNA Access Clean Up 2 with a smudge resistant pen Label a new 96 well HSP plate RAL RNA Access Library with a smudge resistant pen 6 O Part 15049525 Rev B Procedure 10 11 12 13 14 15 16 Centrifuge the RAA plate at 280 x g for 1 minute Remove the adhesive seal from the RAA plate Transfer the entire contents from each well of the RAA plate to the corresponding well of the new 96 well MIDI plate labeled RAC2 Vortex the AMPure XP beads until the beads are well dispersed Add 90 ul well mixed AMPure XP beads to each well of the RAC2 plate containing 50 ul of PCR amplified library Mix thoroughly as follows a Seal the RAC2 plate with a Micro
50. es c 18 cycles of 1 minute incubations starting at 94 C then decreasing 2 C per cycle d 58 C for 90 minutes dy NOTE A 90 minute incubation is optimal for hybridization Hybridizing longer than 2 hours results in a high degree of non specific binding Protocol Label a new 96 well HSP plate RAH1 RNA Access Hyb 1 with a smudge resistant pen Pool Libraries Combine 200 ng of each DNA library for pooling NOTE The TruSeq RNA Access contains enough of each reagent for 4 plex pooling Pooling at less than 4 plex results in the inability to process the number of samples supported by the kit Illumina does not recommend pooling more than 4 samples Pooling an odd number of samples does not affect data For example for 5 samples you can pool 2 samples and 3 samples or 4 samples and 1 sample Table 3 DNA Libraries for Enrichment Library Pool Total DNA Library Complexity Mass ng 1 plex 200 2 plex 400 3 plex 600 4 plex 800 A O Part 15049525 Rev B Procedure 1 3 4 If the total volume is greater than 45 ul concentrate the pooled sample Use either a vacuum concentrator or Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa according to manufacturer instructions If you are using a vacuum concentrator Illumina recommends concentrating samples with a no heat and medium drying rate setting If you are using an Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa it is not required to pre rinse the d
51. evice before use Most of the volume filters through in 5 minutes but up to 30 minutes can be required depending on the starting volume If the pooled sample volume after concentrating is less than 45 ul bring the volume up to 45 ul with Resuspension Buffer Thoroughly vortex the Capture Target Buffer 3 tube until the solution is completely resuspended Visually make sure that no crystal structures are present NOTE If crystals and cloudiness are observed vortex the Capture Target Buffer 3 tube until it is dear Add the following reagents in the order listed to each well of the new 96 well HSP plate labeled RAHI Reagent Volume ul DNA library sample or library pool from TSP1 plate 45 Capture Target Buffer 3 50 Coding Exome Oligos 5 Total Volume per Sample 100 Mix thoroughly as follows a Seal the RAHI plate with a Microseal B adhesive seal Make sure that the plate is tightly sealed to prevent potential evaporation Use an adhesive seal roller to apply force to the seal and make sure that the seal is secured b Shake the RAHI plate on a microplate shaker at 1200 rpm for 1 minute Centrifuge the RAHI plate at 280 x g for 1 minute TruSeq RNA Access Library Prep Guide 41 uonezipuq AH 1814 Protocol 5 Place the sealed RAHI plate on the pre programmed thermal cycler Close the lid then select and run the RNA HYB program a Choose the pre heat lid option and set to 100 C b 95 C for 10 minutes c 18 cycles of 1 minute
52. f pre enrichment sample pooling and the size of the oligonucleotide set Do not add or reduce the cycles of PCR because it can compromise data quality Protocol Procedure Add 5 ul PCR Primer Cocktail to each well of the RAA plate Add 20 ul Enhanced PCR Mix to each well of the RAA plate N 3 Mix thoroughly as follows a Seal the RAA plate with a Microseal A film Use an adhesive seal roller to apply force to the film and make sure that the film is secured b Shake the RAA plate on a microplate shaker at 1200 rpm for 1 minute 4 Centrifuge the RAA plate at 280 x g for 1 minute 5 Place the sealed RAA plate on the pre programmed thermal cycler Close the lid then select and run the EPM AMP program a Choose the pre heat lid option and set to 100 C b 98 C for 30 seconds c 10 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds d 72 C for 5 minutes e Hold at 10 C 5 8 Part 15049525 Rev B e SAFESTOPPING POINT 1 If you do not plan to proceed immediately to Second PCR Clean Up on page 60 the RAA plate can remain on the thermal cycler overnight If you are stopping replace the Microseal A film with a Microseal B adhesive seal and store the RAA plate at 2 C to 8 C for up to two days uoneoyiduuy YOd puooes TruSeqRNA Access Library Prep Guide D 9 Q Second PCR Clean Up 2 O This process uses AMPure XP beads to purify the enriched library and remove unwanted CL products Consumabl
53. he ALP plate to the corresponding well of the new MIDI plate labeled CAP Take care not to disturb the beads Vortex the AMPure XP beads until they are well dispersed TruSeq RNA Access Library Prep Guide p 9 Protocol 30 21 22 23 24 29 26 27 28 29 30 31 32 33 34 35 Add 50 ul mixed AMPure XP beads to each well of the CAP plate for a second cleanup Mix thoroughly as follows a Seal the CAP plate with a Microseal B adhesive seal b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CAP plate at room temperature for 5 minutes Centrifuge the CAP plate at 280 x g for 1 minute Remove the adhesive seal from the CAP plate Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 95 ul of supernatant from each well of the CAP plate Take care not to disturb the beads NOTE Leave the CAP plate on the magnetic stand while performing the following 80 EtOH wash steps 27 29 With the CAP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well Take care not to disturb the beads Incubate the CAP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 27 and 28 one time for a total of two 80 EtOH washes With the CAP plate on the magnetic stand let the samples air dry at roo
54. he scope of research use purposes iii any use of this Product not in accordance with this Products Specifications or Documentation or iv any Excluded Claim TruSeqRNA Access Library Prep Guide V Conditions to Indemnification Obligations The parties indemnification obligations are conditioned upon the party seeking indemnification i promptly notifying the other party in writing of such claim or action ii giving the other party exclusive control and authority over the defense and settlement of such claim or action iii not admitting infringement of any intellectual property right without prior written consent of the other party iv not entering into any settlement or compromise of any such claim or action without the other party s prior written consent and v providing reasonable assistance to the other party in the defense of the claim or action provided that the party reimburses the indemnified party for its reasonable out of pocket expenses incurred in providing such assistance Third Party Goods and Indemnification Illumina has no indemnification obligations with respect to any goods originating from a third party and supplied to Purchaser Third party goods are those that are labeled or branded with a third party s name Purchaser s indemnification rights if any with respect to third party goods shall be pursuant to the original manufacturer s or licensor s indemnity Upon written request Illumina will attempt to pass through such
55. ia deea aa Introduction aaa Library Prep Workflow eeennnn III Prepare Adapter Setup 0 0 0000 cc cece eee eee eee cece ee Fragment RNA ee Synthesize First Strand cDNA 000000000eee eee eee Synthesize Second Strand cDNA 22 2220 2200 Adenylate 3 Ends 2 222 222 2000 Ligate Adapters oo First PCR Amplification 2 2 0 0 0 0 0 0 ccc ccccecccccccccceeeeeees Validate Library nnn First Hybridization ee First Capture aaa Second Hybridization 20 0 2 022 e eee eee eee eee Second Capture 000 22 oo Capture Sample Clean Up oo Second PCR Amplification 0 222 222 ccc eee e cece eeeeeeees Second PCR Clean Up 2 2 2 2 22 2 cece oa Validate Library 1 0 0 0 eee cece cece nnne Appendix A Supporting Information i Introduction aaa ACIODVITIS e ace ze oo Ste ori lee Ulsa a aaa et atatea E Kit Contents c esee eee 0 a i n i aaa Consumables and Equipment eee TruSeq RNA Access Library Prep Kit Indexed Adapter Sequences TruSeq RNA Access Library Prep Guide Technical Assistance Part 15049525 Rev B Overview Introduction RNA Input Recommendations aaa Additional Resources TruSeq RNA Access Library Prep Guide AT i Char GC dick rp secas gei 3 aca Paza p E eze Ln fl LJe1deuo Overview Introduction
56. ibrary Prep protocol has been optimized and validated using the items listed Comparable performance is not guaranteed when using alternate consumables and equipment Table 6 User Supplied Consumables Consumable 1 5 ml RNase DNase free non sticky tubes 1 7 ml microcentrifuge tubes 10 ul barrier pipette tips 10 ul multichannel pipettes 10 ul single channel pipettes 20 ul barrier pipette tips 20 ul multichannel pipettes 20 ul single channel pipettes 200 ul barrier pipette tips 200 ul multichannel pipettes 200 ul single channel pipettes 1000 ul barrier pipette tips 1000 ul multichannel pipettes 1000 ul single channel pipettes Supplier Life Technologies part AM12450 General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier General lab supplier Part 15049525 Rev B Consumable 96 well flat clear bottom black microplates Note Used when quantifying samples with a SpectraMax M5 spectrofluorometer 96 well storage plates round well 0 8 ml MIDI plate Adhesive seal roller Agencourt AMPure XP 60 ml kit Aluminum foil Conical centrifuge tubes 15 ml or 50 ml Ethanol 200 proof absolute for molecular biology 500 ml Hard Shell 96 well PCR Plates HSP plate Microseal A film Microsea
57. illumina TruSeq RNA Access Library Prep Guide ACGAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AATGATAACAGTAACACACTTCTGTTAACCT TAAGATTACTTGT TGATCCACTGAT TCAACGTACCG TATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTAA ete GAT III PC AGA fa INI ME ES RT CAZ TA te ci TAR GA CGT TCR RAGAT ined ental ACCATTAAGAGCTACCGTCTTCTGT T PASSIN RT ACI GAT OCAT ILOR ACCGTAACGAAC AAAAGAATGATAACAGTAACACACTTCTGT TAACCTTAAGAT T ATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAA TTGAGACTAAATAT TAACGTACCAT TAAGAGCTACCGT AAIGATAAGAGAACACACTICTGITAACCT IAAGATTAGT I GATGCAGT GAT TCAAGGTAGOGTAACGAACGIATGAAT GAGAC TAAATAT TAACGIAGCAT TAAGAGCTACOGTCI TCTGT TAACC IAAGATIACT GAT CCAU GAT GAACGIA AAT TGAGAC TAAATAT TAACGTTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG T PORT Ina an AI JAI CARI TEA GA SIE gg EIN GTA GA TTAAGAGCTTCTGTTAACCT TAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAC CGTATCAATTGAGAC TAAATAT TAACGTACT TAACCT TAAGAT TACT TGATCCACTGATTCAACGTACCGTAACGAACG TCTTC TGT TAACC T TAAGAT TAC TT GATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGAC TAACGACGAAA GAGAC TAAATAT TAACGTACCAT TAAGAGCTACAACCT TAAGAT TACT TGATCCACT GAT TCAACGTACCGTAACGAACGTAT CAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TACCGTGCAACGACGAAAAGAAT GATAACAGTAACACACT DAL GA AAC TAG CACI Te OLTRE LIAC a TGA AG GAT EAAS AGER MAREA CITATA ALT GAGA AMATEURS AS AT
58. ing Information to confirm your kit contents and make sure that you have obtained all of the requisite equipment and consumables Part 15049525 Rev B Library Prep Workflow The following illustrates the processes of the TruSeq RNA Access Library Prep protocol to prepare templates using 24 indexed adapter tubes Figure o TruSeq RNA Access Library Prep Workflow Prepare Adapter Setup gt 20 ng Degraded RNA TruSeqRNA Access Library Prep Guide 10 ng Total RNA or Fragment RNA Consumables EPH Water Plates DFP First Strand cDNA Synthesis Consumables FSA SuperScript Il Plate DFP Second Strand cDNA Synthesis Consumables AMPure XP Beads EtOH RSB SMM Plate CCP ALP 3 Adenylate 3 Ends Consumables RSB Plate ALP Ligate Adapters Consumables AMPure XP Beads EtOH LIG RNA Adapters RSB STL Plates CAP PCR 1 First PCR Amplification Consumables AMPure XP Beads EtOH PMM PPC RSB Plate CPP TSP1 x Validate Library 1 First Hybridization Consumables CEX CT3 Plate RAHI First Capture Consumables EE1 ET2 EWS HP3 SMB Plate RAW RAH2 Second Hybridization Consumables CEX CT3 RSB Plate RAH2 Second Capture Consumables EE1 ET2 EWS HP3 SMB Plate RACI RAW2 Capture Sample Clean Up Consumables AMPure XP Beads RSB Plate RAA 1 Second PCR Amplification Consumables EPM PPC Plate RAA Second PCR Cle
59. isturbing the beads Let the RACI plate stand at room temperature for 5 minutes to dry on the magnetic stand Remove the RACI plate from the magnetic stand Add 27 5 ul Resuspension Buffer to each well of the RACI plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the RACI plate with a Microseal B adhesive seal b Shake the RACI plate on a microplate shaker at 1800 rpm for 1 minute Incubate the RACI plate at room temperature for 2 minutes Centrifuge the RACI plate at 280 x g for 1 minute Remove the adhesive seal from the RACI plate Place the RACI plate on the magnetic stand for 2 minutes or until the liquid is clear TruSeq RNA Access Library Prep Guide 5 5 dN uee o ajdwes anides Protocol 22 Transfer 25 ul of clear supernatant from each well of the RACI plate to the corresponding well of the new HSP plate labeled RAA Take care not to disturb the beads 4 NOTE Illumina recommends using a 20 ul single channel or multichannel pipette set to 12 5 ul to perform two consecutive transfers of 12 5 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads SAFE STOPPING POINT If you do not plan to proceed immediately to Second PCR Amplification on page 57 you can safely stop the protocol here If you are stopping seal the RAA plate with a Microseal B adhesive seal and store it at 25 C to 15 C for up to 7 days D
60. ite www illumina com Email techsupport illumina com Table 11 Illumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Austria 0800 296575 Netherlands 0800 0223859 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 Other countries 44 1799 534000 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at support illumina com sds ilmn Product Documentation Product documentation in PDF is available for download from the Illumina website Go to support illumina com select a product then click Documentation amp Literature TruSeq RNA Access Library Prep Guide 8 5 9JUB SISSY EOIUYD AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACC SATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGT TGATCCACTGATTCAACGTACCGTATCAAT TGAGAC TAAATAT TAACGTACCAT TAAGAGCTACCGTCTTCTGITAACCTTAAGAT TACT TGATCCACTGAT TCAACGTACCGT PATATE CEDAT RO A OT STAT AAT A TAG e AC CAT TANGO IR CICI GT PACTI IAA GALIA GAT CA TOAT TEAMO TCR TAA IAAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT TAACGTACCAT TAAGAGCTACC
61. l B adhesive seals Nuclease free ultra pure water One of the following for library quality control Standard Sensitivity NGS Fragment Analysis Kit 1 6000 bp 500 samples DNA 1000 Kit RNase DNase free eight tube strips and caps RNase DNase free multichannel reagent reservoirs disposable RNaseZap to decontaminate surfaces TruSeq RNA Access Library Prep Guide Supplier Corning part 4 3904 Fisher Scientific part AB 0859 General lab supplier Beckman Coulter part A63881 A63880 General lab supplier General lab supplier Sigma Aldrich part E7023 Bio Rad part HSP 9601 Bio Rad part MSA 5001 Bio Rad part MSB 1001 General lab supplier Advanced Analytical Technologies part DNF 473 0500 Agilent Technologies part 5067 1504 General lab supplier VWR part 4 89094 658 General lab supplier f jueuudinb3 pue se qeuunsuo Supporting Information TO Consumable SuperScript II Reverse Transcriptase Tris HCl 10 mM pH8 5 Tween 20 Optional 96 well 2 ml deep well plates to aliquot reagents Optional Amicon Ultra 0 5 centrifugal filter unit 0 5 ml 30 kDa Note Used to concentrate a pooled library Another option is to use a vacuum concentrator Optional One of the following for library quality assessment Standard Sensitivity NGS Fragment Analysis Kit 1 6000 bp 500 samples High Sensitivity DNA Kit Table 7 User Supplied Equip
62. l of the liquid is transferred without disturbing the beads Add 4 ul Elute Target Buffer 2 to each well of the RAH2 plate containing samples to neutralize the elution Part 15049525 Rev B 10 Mix thoroughly as follows a Seal the RAH2 plate with a Microseal B adhesive seal b Shake the RAH2 plate on a microplate shaker at 1200 rpm for 1 minute 11 Centrifuge the RAH2 plate at 280 x g for 1 minute 12 Store the remaining reagents as follows a Place the Elute Target Buffer 2 and Streptavidin Magnetic Beads tubes in 2 C to 8 C storage b Place the 2N NaOH Enrichment Elution Buffer 1 and Enrichment Wash Solution tubes in 25 C to 15 C storage c Discard any remaining elution pre mix SAFESTOPPING POINT 1 If you do not plan to proceed immediately to Second Hybridization on page 48 you can safely stop the protocol here If you are stopping seal the RAH2 plate with a Microseal B adhesive seal and store it at 25 C to 15 C for up to 7 days TruSeq RNA Access Library Prep Guide AT 391n1de 18 114 Protocol Second Hybridization Procedure 48 This process combines the eluted DNA library from the first enrichment round with additional capture probes to targeted regions of interest This second hybridization is required to ensure high specificity of the captured regions Consumables Item Quantity Storage Supplied By Capture Target Buffer 3 CT3 1 tube 25 C to 15 C Illumina Coding Exome Oligos CEX 1 tube
63. le seal b Shake the PCR plate on a microplate shaker at 1600 rpm for 20 seconds 3 Centrifuge the PCR plate at 280 x g for 1 minute 4 Return the PCR Primer Cocktail and Enhanced PCR Mix tubes to 25 C to 15 C storage Amp PCR 1 Place the sealed PCR plate on the pre programmed thermal cycler Close the lid then select and run PCR to amplify the plate a Choose the pre heat lid option and set to 100 C b 98 C for 30 seconds c 15 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds d 72 C for 5 minutes e Hold at 4 C Clean Up PCR 1 Remove the adhesive seal from the PCR plate 34 Part 15049525 Rev B o N A OF 10 11 12 13 14 15 16 17 Vortex the AMPure XP beads for at least 1 minute or until they are well dispersed Add 50 ul mixed AMPure XP beads to each well of the new MIDI plate labeled CPP Transfer the entire contents from each well of the PCR plate to the corresponding well of the CPP plate containing 50 ul mixed AMPure XP beads Mix thoroughly as follows a Seal the CPP plate with a Microseal B adhesive seal b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CPP plate at room temperature for 5 minutes Centrifuge the CPP plate at 280 x g for 1 minute Remove the adhesive seal from the CPP plate Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear Remove and discard 95 ul of the supernata
64. lication Specific IP and Core IP are separate non overlapping subsets of all Illumina owned or controlled intellectual property By way of non limiting example Illumina intellectual property rights for specific diagnostic methods for specific forensic methods or for specific nucleic acid biomarkers sequences or combinations of biomarkers or sequences are examples of Application Specific IP Consumable s means Illumina branded reagents and consumable Part 15049525 Rev B items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Illumina branded instruments accessories or peripherals Illumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hard ware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Illumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to additional terms
65. m 2 C to 8 C storage and bring them to room temperature Label a new 96 well HSP plate RAA RNA Access Amplification with a smudge resistant pen Remove the adhesive seal from the RACI plate Vortex the AMPure XP beads tube until the beads are well dispersed then add 45 ul well mixed AMPure XP beads to each well of the RACI plate Mix thoroughly as follows a Seal the RACI plate with a Microseal B adhesive seal b Shake the RACI plate on a microplate shaker at 1800 rpm for 1 minute Incubate the RACI plate at room temperature for 5 minutes Centrifuge the RACI plate at 280 x g for 1 minute Part 15049525 Rev B 10 11 12 13 14 15 16 17 18 19 20 21 Remove the adhesive seal from the RACI plate Place the RACI plate on the magnetic stand for 2 minutes or until the liquid is clear Remove and discard all of the supernatant from each well of the RACI plate s NOTE f Leave the RACI plate on the magnetic stand while performing the following 80 EtOH wash steps 9 12 With the RACI plate on the magnetic stand slowly add 200 ul freshly made 80 EtOH to each well without disturbing the beads Let the RACI plate stand at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the RACI plate Repeat steps 9 11 one time for a total of two 80 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the RACI plate without d
66. m temperature for 5 minutes Remove the CAP plate from the magnetic stand Add 22 5 ul Resuspension Buffer to each well of the CAP plate Mix thoroughly as follows a Seal the CAP plate with a Microseal B adhesive seal b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CAP plate at room temperature for 2 minutes Centrifuge the CAP plate at 280 x g for 1 minute Remove the adhesive seal from the CAP plate Part 15049525 Rev B 36 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear 37 Transfer 20 ul of supernatant from each well of the CAP plate to the corresponding well of the new HSP plate labeled PCR Take care not to disturb the beads d SAFESTOPPING POINT 1 If you do not plan to proceed immediately to First PCR Amplification on page 32 you can safely stop the protocol here If you are stopping seal the PCR plate with a Microseal B adhesive seal and store at 25 C to 15 C for up to 7 days TruSeq RNA Access Library Prep Guide 31 sJo1depy e1e6r Protocol First PCR Amplification 32 This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library The PCR is performed with a PCR Primer Cocktail that anneals to the ends of the adapters Minimize the number of PCR cycles to avoid skewing the representation of the library t NOTE i
67. ment Equipment DNA Engine Multi Bay Thermal Cycler See Thermal Cyclers on page 81 Fluorometric quantitation with dsDNA binding dye reagents Supplier Invitrogen part 18064 014 General lab supplier Sigma part P7949 Thomson Instrument Company part 951652 Millipore part UFC503008 Advanced Analytical Technologies part DNF 473 0500 Agilent Technologies part 4067 4626 Supplier Bio Rad part PTC 0240G Or e PTC 0220G with Alpha Unit ALS 1296GC General lab supplier Part 15049525 Rev B Equipment One of the following Fragment Analyzer Automated CE System 2100 Bioanalyzer Desktop System High Speed Microplate Shaker Magnetic stand 96 Microcentrifuge Microplate centrifuge MIDI plate insert for heating system Note Two inserts are recommended to support successive heating procedures TruSeq RNA Access Library Prep Guide Supplier Advanced Analytical Technologies part FSv2 CE2 or FSv2 CE10 Agilent Technologies part G2940CA VWR catalog 13500 890 110 V 120 V or e 14216 214 230 V Life Technologies part AM10027 General lab supplier General lab supplier Illumina catalog BD 60 601 T9 jueuudinb3 pue se qeuunsuo Supporting Information Equipment One of the following Note Two systems are recommended to support successive heating procedures SciGene TruTemp Heating System Hybex Microsa
68. mperature for 5 minutes to make sure that all of the beads are bound to the side of the wells 8 Remove and discard 135 ul supernatant from each well of the CCP plate 4 NOTE Leave the CCP plate on the magnetic stand while performing the following 80 EtOH wash steps 9 11 9 With the CCP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads 10 Incubate the CCP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well 11 Repeat steps 9 and 10 one time for a total of two 8076 EtOH washes 12 Letthe CCP plate stand at room temperature for 5 minutes to dry and then remove the CCP plate from the magnetic stand 13 Centrifuge the thawed room temperature Resuspension Buffer at 600 x g for 5 seconds 14 Add 17 5 ul Resuspension Buffer to each well of the CCP plate Mix thoroughly as 22 follows a Seal the CCP plate with a Microseal B adhesive seal b Shake the CCP plate on a microplate shaker at 1800 rpm for 2 minutes Part 15049525 Rev B 15 Incubate the CCP plate at room temperature for 2 minutes 16 Centrifuge the CCP plate at 280 x g for 1 minute 17 Remove the adhesive seal from the CCP plate 18 Place the CCP plate on the magnetic stand at room temperature for 5 minutes 19 Transfer 15 ul supernatant ds cDNA from the CCP plate to the new MIDI plate labeled ALP a SAFE STOPPING POINT 1 If you do not plan to proc
69. mple Incubator QuantiFluor dsDNA System or similar fluorometric based DNA quantification system SpectraMax M5 spectrofluorometer or similar fluorometric based DNA quantification system Stroboscope Vortexer Optional Vacuum concentrator Note Used to concentrate a pooled library Another option is to use Amicon Ultra 0 5 centrifugal filter units Supplier Illumina catalog e SC 60 503 115 V Or e SC 60 504 220 V SciGene catalog 4 1057 30 0 115 V Or e 1057 30 2 230 V Promega catalog 4 E2670 Molecular Devices part 0112 0159 General lab supplier General lab supplier General lab supplier Part 15049525 Rev B Thermal Cyclers The following table lists the recommended settings for the Illumina recommended thermal cycler as well as other comparable models If your lab has a thermal cycler that is not listed validate the thermal cycler before performing the TruSeq RNA Access Library Prep protocol Thermal Cycler Bio Rad DNA Engine Tetrad 2 MJ Research DNA Engine Tetrad Eppendorf Mastercycler Pro S TruSeq RNA Access Library Prep Guide Temp Mode Calculated Calculated Gradient S Simulated Tube Lid Temp Heated Constant at 100 C Heated Heated Vessel Type Polypropylene plates and tubes Plate Plate 81 jueuudinb4 pue se qeuunsuo Supporting Information TruSeq RNA Access Library Prep Kit Indexed Adapter Sequences The TruSeq RNA Acces
70. n Magnetic Beads SMB 1 tube 2 C to 8 C Illumina 1 7 ml microcentrifuge tube i 15 to 30 C User 96 well MIDI plates 2 15 C to 30 C User Microseal B adhesive seals 6 TIC tos Use User Preparation Remove the 2N NaOH Enrichment Elution Buffer 1 and Enrichment Wash Solution from 25 C to 15 C storage and thaw at room temperature Remove the Elute Target Buffer 2 and Streptavidin Magnetic Beads from 2 C to 8 C storage and let stand at room temperature Pre heat the microheating system to 50 C Label a new 96 well MIDI plate RAW2 RNA Access Wash 2 with a smudge resistant pen Part 15049525 Rev B Label a new 96 well MIDI plate RACI RNA Access Clean Up 1 with a smudge resistant pen Second Bind 1 Remove the RAH2 plate from the thermal cycler 2 Centrifuge the room temperature RAH2 plate at 280 x g for 1 minute 3 Remove the adhesive seal from the RAH2 plate Take care when removing the seal to avoid spilling the contents of the wells 4 Transfer the entire contents 100 ul from each well of the RAH2 plate to the corresponding well of the new 96 well MIDI plate labeled RAW2 5 Vortex the Streptavidin Magnetic Beads tube until the beads are well dispersed then add 250 ul well mixed Streptavidin Magnetic Beads to the wells of the RAW2 plate 6 Mix thoroughly as follows a Seal the RAW2 plate with a Microseal B adhesive seal b Shake the RAW2 plate on a microplate shaker at 1200 rpm for 5 minutes 7 Let the
71. nded hybridization time makes sure that targeted regions bind to the capture probes thoroughly This process also describes how to combine multiple libraries with different indexes into a single pool before enrichment Consumables Item Quantity Storage Supplied By Capture Target Buffer 3 CT3 1 tube 25 C to 15 C Ilumina Coding Exome Oligos CEX 1 tube 2o CoE Illumina 96 well HSP plate 1 15 C to 30 C User Microseal B adhesive seal 1 IS C tox BOC User RNase DNase free eight tube 2 15 C to 30 C User strips and caps for multi sample processing Optional Amicon Ultra 0 5 1 per pooled sample ISAS 0 HOKE User centrifugal filter unit 0 5 ml 30 kDa Preparation Remove the following from 25 C to 15 C storage and thaw them at room temperature Capture Target Buffer 3 Coding Exome Oligos For multi sample processing Use a multichannel pipette Distribute the Capture Target Buffer 3 and Coding Exome Oligos into separate eight tube strips dispensing equal volumes into each of the wells TruSeq RNA Access Library Prep Guide 3 9 uonezipuq AH 1814 Remove the TSP1 plate from 25 C to 15 C storage if it was stored at the conclusion of Clean Up PCR and thaw on ice Centrifuge the thawed TSP1 plate at 280 x g for 1 minute Remove the adhesive seal from the thawed TSP1 plate Pre program the thermal cycler with the following program and save as RNA HYB a Choose the pre heat lid option and set to 100 C b 95 C for 10 minut
72. nt from each well of the CPP plate NOTE Leave the CPP plate on the magnetic stand while performing the following 80 EtOH wash steps 10 12 With the CPP plate on the magnetic stand add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Incubate the CPP plate at room temperature for 30 seconds and then remove and discard all of the supernatant from each well Take care not to disturb the beads Repeat steps 10 and 11 one time for a total of two 80 EtOH washes With the CPP plate on the magnetic stand let the samples air dry at room temperature for 5 minutes and then remove the plate from the magnetic stand Add 17 5 ul Resuspension Buffer to each well of the CPP plate Make sure the Resuspension Buffer runs over the beads Mix thoroughly as follows a Seal the CPP plate with a Microseal B adhesive seal b Shake the CPP plate on a microplate shaker at 1800 rpm for 2 minutes Incubate the CPP plate at room temperature for 2 minutes Centrifuge the CPP plate at 280 x g for 1 minute Remove the adhesive seal from the CPP plate TruSeq RNA Access Library Prep Guide 3 5 uoneoyiduuv yO d 18414 Protocol 18 Place the CPP plate on the magnetic stand at room temperature for 5 minutes or until the liquid is clear 19 Transfer 15 ul of the clear supernatant from each well of the CPP plate to the corresponding well of the new HSP plate labeled TSP1 SAFESTOPPING POINT If you do not plan to proceed imme
73. ocktail at 600 x g for 5 seconds Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Remove the AMPure XP beads from 2 C to 8 C storage and let stand for at least 30 minutes to bring them to room temperature Remove the PCR plate from 25 C to 15 C storage if it was stored at the conclusion of Clean Up ALP on page 28 Let it thaw at room temperature Centrifuge the thawed PCR plate at 280 x g for 1 minute Remove the adhesive seal from the thawed PCR plate Pre program the thermal cycler with the following program and save as PCR Choose the pre heat lid option and set to 100 C 98 C for 30 seconds 15 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 minutes Hold at 4 C TruSeq RNA Access Library Prep Guide 3 3 uoneoyiduuv HOd 18414 Protocol Label a new 96 well MIDI plate CPP Clean Up PCR Plate with a smudge resistant pen Label a new 96 well HSP plate TSP1 Target Sample Plate with a smudge resistant pen Make PCR 1 Add 5 ul thawed PCR Primer Cocktail to each well of the PCR plate 2 Add25 ul thawed PCR Master Mix to each well of the PCR plate a Seal the PCR plate with a Microseal A film WARNING Follow vendor instructions for applying Microseal A sealing films Improper use could lead to inefficient sealing evaporation of sample or cross contamination or too efficient sealing parts of the seal remain in the well after removing the who
74. on the bench 3 Remove the adhesive seal from the DEP plate 4 Let the DFP plate stand to bring it to room temperature Remove the AMPure XP beads from storage and let stand for at least 30 minutes to bring them to room temperature Review Best Practices for Handling Magnetic Beads See Additional Resources on page 7 for information on how to access TruSeq RNA Access Library Prep Best Practices on the Illumina website Pre heat the thermal cycler to 16 C Choose the thermal cycler pre heat lid option and set the lid to 30 C Label a new 96 well MIDI plate ALP Adapter Ligation Plate with a smudge resistant pen Label a new 96 well MIDI plate CCP cDNA Clean Up Plate with a smudge resistant pen TruSeq RNA Access Library Prep Guide 2 1 YNQ2 pues puooeg aziseyju s Protocol Clean Up DFP 1 Vortex the AMPure XP beads until they are well dispersed 2 Add 90 ul of well mixed AMPure XP beads to each well of the new MIDI plate labeled CCP 3 Transfer the entire contents from each well of the DFP plate to the corresponding well of the CCP plate containing AMPure XP beads Mix thoroughly as follows a Seal the CCP plate with a Microseal B adhesive seal b Shake the CCP plate on a microplate shaker at 1800 rpm for 2 minutes 4 Incubate the CCP plate at room temperature for 5 minutes 5 Centrifuge the CCP plate at 280 x g for 1 minute 6 Remove the adhesive seal from the CCP plate 7 Place the CCP plate on the magnetic stand at room te
75. or any Illumina Infringement Claim to the extent such infringement arises from i the use of this Product in any manner or for any purpose outside the scope of research use purposes ii the use of this Product in any manner not in accordance with its Specifications its Documentation the rights expressly granted to Purchaser hereunder or any breach by Purchaser of these terms and conditions iii the use of this Product in combination with any other products materials or services not supplied by Illumina iv the use of this Product to perform any assay or other process not supplied by Illumina or v Illumina s compliance with specifications or instructions for this Product furnished by or on behalf of Purchaser each of i v is referred to as an Excluded Claim Indemnification by Purchaser Purchaser shall defend indemnify and hold harmless Illumina its affiliates their non affiliate collaborators and development partners that contributed to the development of this Product and their respective officers directors representatives and employees against any claims liabilities damages fines penalties causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii Purchaser s use of this Product outside of t
76. plate with a Microseal B adhesive seal b Shake the RAC2 plate on a microplate shaker at 1800 rpm for 1 minute Incubate the RAC2 plate at room temperature for 2 minutes Centrifuge the RAC2 plate at 280 x g for 1 minute Remove the adhesive seal from the RAC2 plate Place the RAC2 plate on the magnetic stand for 2 minutes or until the liquid is clear Transfer 30 ul of clear supernatant from each well of the RAC2 plate to the corresponding well of the new HSP plate labeled RAL Take care not to disturb the beads NOTE i Ilumina recommends using a 20 ul single channel or multichannel pipette set to 15 ul to perform two consecutive transfers of 15 ul This technique reduces sample loss by making sure that all of the liquid is transferred without disturbing the beads Seal the RAL plate with a Microseal B adhesive seal SAFE STOPPING POINT If you do not plan to proceed immediately to Validate Library on page 63 store the sealed RAL plate at 25 C to 15 C for up to 7 days If the plate is stored for more than 7 days requantify your library to guarantee the accuracy of your enrichment results Part 15049525 Rev B Validate Library Illumina recommends performing the following procedures for quality control analysis and quantification of your enriched library Quantify Libraries To achieve the highest quality data on Illumina sequencing platforms it is important to create optimum cluster densities across every lane of th
77. rop For best performance on samples close to edge of a quality classification err towards the higher end of the input recommendation 4 NOTE For more information see the Evaluating RNA Quality from FFPE Samples tech note for TruSeq RNA Access Library Prep See Additional Resources on page 7 for information on how to download the tech note from the Illumina website Part 15049525 Rev B The following are examples of high medium and low quality FEPE traces and a trace of FFPE quality that is not recommended for use for TruSeq RNA Access Library Prep Figure 2 Example High Quality FFPE DV 77 suonepueuJulooeH 1ndu YNY TruSeq RNA Access Library Prep Guide D Overview Figure 4 Example Low Quality FFPE DV 30 Positive Control Illumina recommends using Agilent Technologies Human UHR total RNA catalog 740000 as a positive control sample for this protocol 6 Part 15049525 Rev B Additional Resources The following resources are available for TruSeq RNA Access Library Prep protocol guidance and sample tracking Access these and other resources on the Illumina website at support illumina com sequencing kits ilmn Then select TruSeq RNA Access Library Prep Kit Support Resource Training Best Practices TruSeq RNA Access Library Prep Experienced User Card and Lab Tracking Form part 15049526 Evaluating RNA Quality from FFPE Samples tech note TruSeq RNA Access Librar
78. s Library Prep Kit contains the following indexed adapter sequences 1 NOTE The index numbering is not contiguous There is no Index 17 24 or 26 The base in parentheses indicates the base for the seventh cycle and is not considered as part of the index sequence Record the index in the sample sheet as only six bases For indexes 13 and above the seventh base in parentheses might not be A which is seen in the seventh cycle of the Index Read For more information on the number of cycles used to sequence the Index Read reference your instrument user guide Table 8 TruSeq RNA Access Library Prep Kit Set A Indexed Adapter Sequences Adapter Sequence Adapter Sequence AR002 CGATGT A ARO013 AGTCAA C AR004 TGACCA A ARO14 AGTTCC G AR005 ACAGTG A ARO15 ATGTCA G ARO006 GCCAAT A ARO16 CEETEE E AROO7 CAGATC A ARO018 GTCCGC A AR012 CTTGTA A AR019 GTGAAA C 8 p Part 15049525 Rev B Table 9 TruSeq RNA Access Library Prep Kit Set B Indexed Adapter Sequences Adapter AR001 AR003 ARO008 AR009 ARO010 ARO011 TruSeq RNA Access Library Prep Guide Sequence Adapter TTAGGC A ARO21 serene XN wem O s Sequence GTGGCC T GTTICG G CGTACG T GAGTGG A ACTGAT A ATICCT T 83 pexepu uy deJg le1qr sse2oy YNY besni 84 Part 15049525 Rev B Technical Assistance For technical assistance contact Illumina Technical Support Table 10 Illumina General Contact Information Illumina Webs
79. seal B adhesive seal b Shake the RAC2 plate on a microplate shaker at 1800 rpm for 1 minute Incubate the RAC2 plate at room temperature for 5 minutes Centrifuge the RAC2 plate at 280 x g for 1 minute Remove the adhesive seal from the RAC2 plate Place the RAC2 plate on the magnetic stand at room temperature for 2 minutes or until the liquid is clear Carefully remove and discard all of the supernatant from each well of the RAC2 plate t NOTE Leave the RAC2 plate on the magnetic stand while performing the following 80 EtOH wash steps 12 15 With the RAC2 plate on the magnetic stand slowly add 200 ul freshly prepared 80 EtOH to each well without disturbing the beads Let the RAC2 plate stand at room temperature for 30 seconds Remove and discard the 80 EtOH from each well of the RAC2 plate Repeat steps 12 14 one time for a total of two 8076 EtOH washes Using a 20 ul single channel or multichannel pipette remove any remaining 80 EtOH from each well of the RAC2 plate without disturbing the beads TruSeq RNA Access Library Prep Guide 61 dn ueso HOd PUODAS Protocol 17 18 19 20 21 22 23 24 25 26 1 62 Let the RAC2 plate stand at room temperature for 5 minutes to dry on the magnetic stand Remove the RAC2 plate from the magnetic stand Add 32 ul Resuspension Buffer to each well of the RAC2 plate Do not touch the beads with the pipette tips Mix thoroughly as follows a Seal the RAC2
80. ss the upgrade was conducted by Illumina at Illumina s facilities in which case the upgraded Hardware shipped to Purchaser comes with a Base Hardware Warranty c Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect negligence accident improper storage or use contrary to the Documentation or Specifications ii improper handling installation maintenance or repair other than if performed by Illumina s personnel iii unauthorized alterations iv Force Majeure events or v use with a third party s good not provided by Illumina unless the Product s Documentation or Specifications expressly state such third party s good is for use with the Product d Procedure for Warranty Coverage In order to be eligible for repair or replacement under this warranty Purchaser must i promptly contact Illumina s support department to report the non conformance ii cooperate with Illumina in confirming or diagnosing the non conformance and iii return this Product transportation charges prepaid to IV Part 15049525 Rev B Illumina following Illumina s instructions or if agreed by Illumina and Purchaser grant Illumina s authorized repair personnel access to this Product in order to confirm the non conformance and make repairs Sole Remedy under Warranty Illumina will at its option repair or replace non conforming Product that it confirms is covered by this warran
81. t High Mix Enhanced PCR Mix Elute Target Buffer 2 Experienced User Card 67 sul uoJov Supporting Information 68 Acronym EWS FEPE FSA HP3 HSP IEM LIG LTE PCR PMM PPC RAC1 RAC2 RAH1 RAH2 RAL RAW1 RAW2 RFU RIN Definition Enrichment Wash Solution Formalin fixed paraffin embedded First Strand Synthesis Act D Mix 2N NaOH Hardshell Plate Ilumina Experiment Manager Ligation Mix Lab Tracking Form Polymerase Chain Reaction PCR Master Mix PCR Primer Cocktail NA Access Amplification Plate NA Access Clean Up Plate 1 NA Access Clean Up Plate 2 NA Access Hyb Plate 1 NA Access Hyb Plate 2 NA Access Library Plate NA Access Wash Plate 1 zE n FA 7A nuu NA Access Wash Plate 2 Relative Fluorescence Unit RNA integrity number Part 15049525 Rev B Acronym rRNA RSB SMB SMM SIUE TSP TruSeq RNA Access Library Prep Guide Definition Ribosomal RNA Resuspension Buffer Streptavidin Magnetic Beads Second Strand Marking Master Mix Stop Ligation Buffer Target Sample Plate 69 suJ uoJov Supporting Information Kit Contents Check to make sure that you have all of the reagents identified in this section before starting the TruSeq RNA Access Library Prep protocol The TruSeq RNA Access Library Prep Kit is available in a Set A and a Set B Each TruSeq RNA Access Library Prep Kit contains enough reagents to prepare up to 48 samples When used together sets A and B
82. tes or until the liquid is clear Remove the adhesive seal from the RAW2 plate Immediately remove and discard all of the supernatant from each well of the RAW2 plate 10 Remove the RAW2 plate from the magnetic stand 11 Repeat steps 2 10 one time for a total of two Enrichment Wash Solution washes Second Elution oc 1 Add the following reagents in the order listed in a new 1 7 ml microcentrifuge tube to create the elution pre mix Multiply each volume by the number of pooled samples being prepared The volumes include an excess amount for processing multiple samples Reagent Volume ul Enrichment Elution Buffer 1 28 5 2N NaOH 1 5 Total volume per enrichment 30 Vortex the elution pre mix tube then add 23 ul of the mix to each well of the RAW2 plate Part 15049525 Rev B 3 Mix thoroughly as follows a Seal the RAW2 plate with a Microseal B adhesive seal b Shake the RAW2 plate on a microplate shaker at 1800 rpm for 2 minutes 4 Let the RAW2 plate stand at room temperature for 2 minutes 5 Centrifuge the RAW2 plate at 280 x g for 1 minute 6 Carefully remove the adhesive seal from the RAW2 plate to avoid spilling the contents of the wells 7 Place the RAW2 plate on the magnetic stand for 2 minutes or until the liquid is clear 8 Transfer 21 ul of clear supernatant from each well of the RAW2 plate to the corresponding well of the new MIDI plate labeled RACI Take care not to disturb the beads y NOTE Illumin
83. the following components at 25 C to 15 C Set A TruSeq RNA Access 12 Index Set A 48 Samples Box 2 part 15052311 Slot Reagent Part Description Resuspension Buffer Elution Primer Fragmentation Mix Ligation Mi Aang Ma Stop Ligation Buffer 15024655 RNA Adapter Index 13 15024656 RNA Adapter Index 14 15024657 RNA Adapter Index 15 15024658 RNA Adapter Index 16 15024660 RNA Adapter Index 18 15024661 RNA Adapter Index 19 15026634 RNA Adapter Index 2 15026636 RNA Adapter Index 4 XO CO ND OT mo PN A Rl RI lO W Part 15049525 Rev B Slot 14 15 16 17 Set B TruSeq RNA Access 12 Index Slot SYO o d OOo mo MY A Rl Rl Rl Re Re NIAN OT A oO N e c Reagent AR005 AR006 AR007 AR012 Reagent RSB EPH LIG ATL SIL AR020 ARO021 AR022 AR023 AR025 AR027 AROO1 AR003 AR008 AR009 ARO10 ARO11 Part 15026637 15026638 15026640 15026645 Part 15026770 15029211 15026773 15012495 15012546 15024662 15024663 15024664 15024665 15024667 15024668 15026633 15026635 15026641 15026642 15026643 15026644 TruSeq RNA Access Library Prep Guide Description RNA Adapter Index 5 RNA Adapter Index 6 RNA Adapter Index 7 RNA Adapter Index 12 Set B 48 Samples Box 2 part 15052312 Description Resuspension Buffer Elution Primer Fragmentation Mix Ligation Mix A Tailing Mix Stop Ligation Buffer NA Adapter Index 20 NA
84. ty Repaired or replaced Consumables come with a 30 day warranty Hardware may be repaired or replaced with functionally equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Illumina will attempt to pass through any such warranty to Purchaser 9 Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Illumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligations Section 9 d below Illumina shall i
85. umina Second Strand Marking Master 1 tube per 48 25 Cto 152 Illumina Mix SMM reactions 96 well MIDI plates 2 15 C to 30 C User AMPure XP beads 90 ul per sample DAC tio IAC User Freshly prepared 8076 ethanol 400 ul per sample 15 C to 30 C User EtOH Microseal B adhesive seals 4 TIC O SUE User RNase DN ase free eight tube 5 15 C to 30 C User strips and caps if using multichannel pipettes RNase DNase free reagent 5 USMS tro SUE User reservoirs if using multichannel pipettes Preparation Remove the Second Strand Marking Master Mix from 25 C to 15 C storage and thaw it at room temperature Remove the Resuspension Buffer from 2 C to 8 C storage and bring it to room temperature Part 15049525 Rev B Add SMM 1 Remove the adhesive seal from the DEP plate 2 Add5 ul of Resuspension Buffer to each well of the DEP plate 3 Centrifuge the thawed Second Strand Marking Master Mix at 600 x g for 5 seconds 4 Add 20 ul of thawed Second Strand Marking Master Mix to each well of the DEP plate Mix thoroughly as follows a Seal the DEP plate with a Microseal B adhesive seal b Shake the DFP plate on a microplate shaker continuously at 1600 rpm for 20 seconds 5 Return the Second Strand Marking Master Mix tube to 25 C to 15 C storage after use Incubate 3 DFP 1 Place the sealed DEP plate on the pre heated thermal cycler Close the lid and incubate at 16 C for 1 hour 2 Remove the DFP plate from the thermal cycler and place it
86. y Prep Guide Description Illustrates elements of the TruSeq RNA Access Library Prep process Viewing these videos is recommended for new and less experienced users before starting library prep Click Training on TruSeq RNA Access Library Prep Kit Support Provides best practices specific to this protocol Review these best practices before starting library prep Topics include Handling Liquids Handling Master Mix Reagents Handling Magnetic Beads Avoiding Cross Contamination Potential DNA Contaminants Temperature Considerations Equipment Click Best Practices on TruSeq RNA Access Library Prep Kit Support Provides protocol instructions but with less detail than what is provided in this user guide New or less experienced users are advised to follow this user guide and not the EUC and LTF Click Documentation amp Literature on TruSeq RNA Access Library Prep Kit Support Provides effectivity profiles for FFPE RNA Click Documentation amp Literature on TruSeq RNA Access Library Prep Kit Support S992JnoseH euonippV Overview Resource TruSeq Sample Preparation Pooling Guide part 15042173 Sequencing Library qPCR Quantification Guide part 4 11322363 Illumina Experiment Manager IEM BaseSpace Description Provides TruSeq pooling guidelines for library prep Review this guide before beginning library preparation Click Documentation amp Literature on TruSeq RNA Access Librar
87. y Prep Kit Support Describes a qPCR method for quantifying sequencing by synthesis SBS libraries generated using the Illumina library prep protocols Click Documentation amp Literature on TruSeq RNA Access Library Prep Kit Support Enables you to create and edit appropriate sample sheets for Illumina sequencing systems and analysis software and record parameters for your sample plate To download the software click Downloads on TruSeq RNA Access Library Prep Kit Support To download the documentation click Documentation amp Literature on TruSeq RNA Access Library Prep Kit Support Sequencing data analysis tool that also enables you to organize samples libraries pools and run in a single environment For more information on BaseSpace see support illumina com sequencing sequencing _ software basespace ilmn Part 15049525 Rev B Protocol Introduction cnc cnc cnc 10 Library Prep Workflow nn e ess esses sess ese 11 Prepare Adapter Sel p noia tt a at a ta 12 Fragment RNA ceo etu cepe es pared NN E coral Lee LUE 13 Synthesize First Strand cDNA esse e eee ere esse rre erret rris 17 Synthesize Second Strand cDNA aaa 20 Adenylate 3 Ends s n cae eat cate dla cada 24 Ligate Adapters RETRO 26 First PCR Amplification aaa aaa 32 Validate Library MERE RR 37 First Hybridization esise noa iure ope os acacia ia E P dedat a aaa 39 First Capture nas 43 Second Hybridization secara iia 48 Second

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