TruSeq RNA Exome. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v00 October 2017

Transcription

1 TrSeq RNA Exome Reference Gide Docment # v00 October 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSeq RNA Exome Reference Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. Illmina, BaseSpace, TrSeq, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Limited Use Label License: This prodct and its se are the sbject of one or more issed and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft, exclsively licensed to New England Biolabs, Inc. and sblicensed to Illmina, Inc. The prchase of this prodct from Illmina, Inc., its affiliates, or its athorized resellers and distribtors conveys to the byer the non-transferable right to se the prchased amont of the prodct and components of the prodct by the byer (whether the byer is an academic or for profit entity). The prchase of this prodct does not convey a license nder any claims in the foregoing patents or patent applications directed to prodcing the prodct. The byer cannot sell or otherwise transfer this prodct or its components to a third party or otherwise se this prodct for the following COMMERCIAL PURPOSES: (1) se of the prodct or its components in manfactring; or (2) se of the prodct or its components for therapetic or prophylactic prposes in hmans or animals. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. ii

3 Table of Contents Chapter 1 Overview 1 Introdction 1 RNA Inpt Recommendations 1 Additional Resorces 4 Chapter 2 Protocol 5 Introdction 5 Tips and Techniqes 5 Library Prep Workflow 7 Prepare for Pooling 8 Fragment RNA 8 Synthesize First Strand cdna 9 Synthesize Second Strand cdna 10 Adenylate 3ʹ Ends 12 Ligate Adapters 12 Enrich DNA Fragments 15 Check Libraries 17 Hybridize Probes 18 Captre Hybridized Probes 20 Perform Second Hybridization 22 Perform Second Captre 23 Clean Up Captred Library 25 Amplify Enriched Library 26 Clean Up Amplified Enriched Library 27 Check Enriched Libraries 28 Appendix A Spporting Information 30 Introdction 30 Prodct Contents 30 Consmables and Eqipment 32 Index Adapter Seqences 34 Acronyms 34 Technical Assistance 36 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. iii

4 Chapter 1 Overview Introdction 1 RNA Inpt Recommendations 1 Additional Resorces 4 Introdction This protocol explains how to convert total RNA into a library of template molecles of known strand origin, and then captre the coding regions of the transcriptome sing reagents for the Illmina TrSeq RNA Exome workflow. The library is sitable for sbseqent clster generation and DNA seqencing. The RNA is fragmented sing divalent cations nder elevated temperatre. cdna is generated from the cleaved RNA fragments sing random priming dring first and second strand synthesis. Then, seqencing adapters are ligated to the reslting doble-stranded cdna fragments. The coding regions of the transcriptome are captred from this library sing seqence-specific probes to create the final library. This library prep protocol offers: High data qality even from degraded or FFPE-derived RNA samples Inpt reqirement as low as 10 ng for fresh/frozen samples and 20 ng for FFPE samples Uniform captre of the coding transcriptome, redcing seqencing reqirement while maintaining discovery power Up to 24 niqe indexes and 4-plex pre-enrichment pooling for the most efficient se of yor seqencing read bdget Strand information on RNA transcripts High throghpt atomation-friendly procedres RNA Inpt Recommendations Total RNA Inpt The protocol is optimized for ng of hman total RNA. Lower amonts might reslt in low yield and inefficient ligation. The protocol has been tested sing 10 ng of high-qality niversal hman reference total RNA as inpt. Use of RNA from other tisses or lower qality RNA might reqire frther optimization to determine the inpt amont. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 1

5 TrSeq RNA Exome Reference Gide Determine the qality of the RNA starting material. The fragmentation conditions are optimized for high-qality RNA. Use the Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity RNA Analysis Kit to determine the qality of yor starting material. The following figre shows a Universal Hman Reference (UHR) starting RNA Bioanalyzer trace. Figre 1 Starting RNA Bioanalyzer Trace Degraded or FFPE RNAs are shorter than fll length RNA. DNA contamination cases an nderestimation of the amont of RNA sed. If starting with FFPE RNA, the sample inpt amont is based on sample qality. Use the percentage of RNA fragments > 200 nt fragment distribtion vale (DV200) as a reliable determinant of FFPE RNA qality. Table 1 FFPE RNA Inpt Recommendations Qality DV200 Inpt Reqirement Per Reaction High > 70% 20 ng Medim 50 70% ng Low 30 50% ng Too degraded < 30% Not recommended For sccessfl library prep, se an RNA isolation method that incldes a reverse-crosslinking step and DNase1 treatment, sch as the QIAGEN RNeasy FFPE Kit or QIAGEN AllPrep DNA/RNA FFPE Kit. Use NanoDrop to determine FFPE RNA concentration. For samples that border a qality classification, error towards the higher end of the inpt recommendation. NOTE For more information, see the Evalating RNA Qality from FFPE Samples tech note for the TrSeq RNA Exome workflow. See Additional Resorces on page 1 for information on how to download the tech note from the Illmina website. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 2

6 TrSeq RNA Exome Reference Gide The following are examples of high, medim, and low qality FFPE traces, pls a trace of FFPE qality that is not recommended for se with TrSeq RNA Exome workflow. Figre 2 Example: High Qality FFPE (DV200 = 77%) Figre 3 Example: Medim Qality FFPE (DV200 = 55%) Figre 4 Example: Low Qality FFPE (DV200 = 30%) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 3

7 TrSeq RNA Exome Reference Gide Figre 5 Example: FFPE Qality Not Recommended for Use (DV200 = 8%) Positive Control Use Agilent Technologies Hman UHR total RNA (catalog # ) as a positive control sample for this protocol. Additional Resorces The following docmentation is available for download from the Illmina website. Resorce TrSeq RNA Exome Checklist (docment # ) Evalating RNA Qality from FFPE Samples tech note TrSeq Library Prep Pooling Gide (docment # ) Illmina Adapter Seqences (docment # ) Seqencing Library qpcr Qantification Gide (docment # ) Illmina Experiment Manager Gide (docment # ) and IEM TrSeq DNA, RNA, or ChIP Qick Reference Card (docment # ) BaseSpace help (help.basespace.illmina.com) Local Rn Manager Software Gide (docment # ) Description Provides a checklist of the protocol steps. The checklist is intended for experienced sers. Provides effectivity profiles for FFPE RNA. Provides TrSeq pooling gidelines for preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Review this gide before beginning library preparation. Provides the ncleotide seqences that comprise Illmina oligoncleotides sed in Illmina seqencing technologies. Describes a qpcr method for qantifying seqencing by synthesis (SBS) libraries generated sing the Illmina library prep protocols. Provide information abot creating and editing appropriate sample sheets for Illmina seqencing systems and analysis software and record parameters for yor sample plate. Provides information abot the BaseSpace Seqence Hb seqencing data analysis tool that also enables yo to organize samples, libraries, pools, and seqencing rns in a single environment. Provides an overview of the Local Rn Manager (LRM) software, instrctions for sing software featres, and instrctions for installing analysis modles on the instrment compter. Visit the TrSeq RNA Exome workflow spport page on the Illmina website for access to reqirements and compatibility, additional docmentation, software downloads, online training, freqently asked qestions, and best practices. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 4

8 Chapter 2 Protocol Introdction 5 Tips and Techniqes 5 Library Prep Workflow 7 Prepare for Pooling 8 Fragment RNA 8 Synthesize First Strand cdna 9 Synthesize Second Strand cdna 10 Adenylate 3ʹ Ends 12 Ligate Adapters 12 Enrich DNA Fragments 15 Check Libraries 17 Hybridize Probes 18 Captre Hybridized Probes 20 Perform Second Hybridization 22 Perform Second Captre 23 Clean Up Captred Library 25 Amplify Enriched Library 26 Clean Up Amplified Enriched Library 27 Check Enriched Libraries 28 Introdction This section describes the TrSeq RNA Exome workflow protocol. Follow the protocols in the order shown, sing the specified volmes and incbation parameters. Review Best Practices before proceeding. See Additional Resorces on page 1 for information on how to access TrSeq RNA Exome workflow Best Practices on the Illmina website. Before proceeding, confirm kit contents and make sre that yo have the reqired eqipment and consmables. For more information, see Spporting Information on page 30. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. When adding adapters or primers, change tips between each row and each colmn. Remove nsed index adapter tbes from the working area. Sealing the Plate Always seal the 96-well plate before the following steps in the protocol: Shaking steps Vortexing steps Centrifge steps Thermal cycling steps Apply the adhesive seal to cover the plate, and seal with a rbber roller. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 5

9 TrSeq RNA Exome Reference Gide Microseal 'B' adhesive seals are effective at -40 C to 110 C, and sitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifging, and long-term storage. Microseal 'A' adhesive film is sed for thermal cycling steps to prevent evaporation. Plate Transfers When transferring volmes between plates, transfer the specified volme from each well of a plate to the corresponding well of the other plate. Centrifgation Centrifge at any step in the procedre to consolidate liqid or beads in the bottom of the well, and to prevent sample loss. Handling Beads Do not freeze beads. Pipette bead sspensions slowly. Before se, allow the beads to come to room temperatre. Immediately before se, vortex the beads ntil they are well dispersed. The color of the liqid mst appear homogeneos. Vortex throghot protocol as necessary to keep homogenos. If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the liqid is clear (~2 mintes). When washing beads: Use the specified magnetic stand for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnetic stand ntil the instrctions specify to remove it. Do not agitate the plate while it is on the magnetic stand. Do not distrb the bead pellet. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 6

10 TrSeq RNA Exome Reference Gide Library Prep Workflow Figre 6 TrSeq RNA Exome workflow Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 7

11 TrSeq RNA Exome Reference Gide Prepare for Pooling When pooling samples for seqencing, se IEM, LRM, or BaseSpace Prep Tab to record information abot yor samples before beginning library preparation. Use IEM to create and edit sample sheets for Illmina seqencing systems and analysis software. Use LRM and BaseSpace Prep Tab to organize samples, libraries, pools, and a rn for Illmina seqencing systems and analysis software. Inclde a common index in each colmn. A common index facilitates pipetting operations when dispensing index adapters and pooling indexed libraries. Review the planning steps in the TrSeq Library Prep Pooling Gide (docment # ) when preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Fragment RNA This process fragments and primes RNA for cdna synthesis. NOTE If starting with FFPE RNA, see Total RNA Inpt on page 1 for more information. Do not perform the incbation steps 5 6 in this procedre. Consmables EPH (Elte, Prime, Fragment High Mix) RSB (Resspension Bffer) Total RNA (10 ng fresh/frozen RNA per reaction or ng FFPE RNA per reaction) Ultrapre Water 96-well Hard-Shell 0.3 ml PCR plate Microseal 'B' adhesive seal Preparation 1 Prepare the following consmables. Item Storage Instrctions EPH -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB -25 C to -15 C Thaw at room temperatre. Store at 2 C to 8 C after the initial thaw. 2 Save the following Eltion 2-Frag-Prime program on the thermal cycler. Choose the preheat lid option and set to 100 C 94 C for 8 mintes Hold at 4 C Each well contains 17 µl 3 Set the centrifge to 15 C to 25 C. 4 Label a new Hard-Shell PCR plate DFP with a marker. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 8

12 TrSeq RNA Exome Reference Gide Procedre 1 Dilte the total RNA in nclease-free ltrapre water to a final volme of 8.5 μl in each well of the DFP plate. 2 Add 8.5 µl EPH to each well. 3 Apply the seal and shake at 1600 rpm for 20 seconds. 4 Apply the seal and centrifge at 280 g for 1 minte. WARNING If starting with FFPE RNA, do not perform the following incbation procedre. Proceed immediately to Synthesize First Strand cdna on page 9. 5 Place on the preprogrammed thermal cycler and rn the Eltion 2-Frag-Prime program. 6 Centrifge briefly. Synthesize First Strand cdna This process reverse transcribes the cleaved RNA fragments primed with random hexamers into first strand cdna. The addition of Actinomycin D to the FSA (First Strand Synthesis Act D Mix) prevents sprios DNAdependent synthesis, while allowing RNA-dependent synthesis, and improving strand specificity. Consmables FSA (First Strand Synthesis Act D Mix) SperScript II Reverse Transcriptase Microseal 'B' adhesive seals WARNING FSA contains Actinomycin D, a toxin. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any nsed contents in accordance with the governmental safety standards for yor region. See the safety data sheet (SDS) for environmental, health, and safety information. For more information, see Technical Assistance on page 36. Preparation 1 Prepare the following consmables. Item Storage Instrctions FSA -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. 2 Save the following Synthesize 1st Strand program on the thermal cycler: Choose the preheat lid option and set to 100 C 25 C for 10 mintes 42 C for 15 mintes 70 C for 15 mintes Hold at 4 C Each well contains 25 µl Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 9

13 TrSeq RNA Exome Reference Gide Procedre 1 Centrifge FSA at 600 g for 5 seconds. 2 Add 50 µl SperScript II to FSA. Pipette to mix. Then apply the seal and centrifge briefly. Label the FSA tbe to indicate that SperScript II has been added. NOTE If yo are not sing the entire contents of FSA, add SperScript II at a ratio of 1 µl SperScript II to 9 µl FSA. The mixtre can be sed for sbseqent experiments. For more than 6 freeze-thaw cycles, prepare 10 µl aliqots and store at -25 C to -15 C. 3 Add 8 µl SperScript II and FSA mixtre to each well. 4 Apply the seal and shake at 1600 rpm for 20 seconds. 5 Place on the preprogrammed thermal cycler and rn the Synthesize 1st Strand program. Synthesize Second Strand cdna This process removes the RNA template, synthesizes a replacement strand, and incorporates dutp in place of dttp to generate ds cdna. The incorporation of dutp qenches the second strand dring amplification. Magnetic beads separate the ds cdna from the second strand reaction mix. The reslt is blnt-ended cdna. Consmables RSB (Resspension Bffer) SMM (Second Strand Marking Master Mix) AMPre XP beads Freshly prepared 80% ethanol (EtOH) 96-well midi plates (2) Microseal 'B' adhesive seals Abot Reagents Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions SMM -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 10

14 TrSeq RNA Exome Reference Gide 2 Choose the thermal cycler preheat lid option and set the lid to 30 C 3 Preheat the thermal cycler to 16 C. 4 Label a new Hard-Shell PCR plate ALP with a marker. 5 Label a new midi plate CCP with a marker. Procedre Add SMM 1 Add 5 µl RSB to each well. 2 Centrifge SMM at 600 g for 5 seconds. 3 Add 20 µl SMM to each well. 4 Apply the seal and shake at 1600 rpm for 20 seconds. 5 Apply the seal and centrifge at 280 g for 1 minte. 6 Place on the preprogrammed thermal cycler and incbate at 16 C for 1 hor. Each well contains 50 µl. 7 Place on the bench and let stand to bring to room temperatre. Prify cdna 1 Add 90 µl AMPre XP beads to the CCP plate. 2 Transfer all from the DFP plate to the corresponding well of the CCP plate. 3 Apply the seal and shake at 1800 rpm for 2 mintes. 4 Incbate at room temperatre for 5 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 7 Remove and discard 135 µl spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 5 mintes. 11 Remove from the magnetic stand. 12 Centrifge RSB at 600 g for 5 seconds. 13 Add 17.5 µl RSB to each well. 14 Apply the seal and shake at 1800 rpm for 2 mintes. 15 Incbate at room temperatre for 2 mintes. 16 Centrifge at 280 g for 1 minte. 17 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 11

15 TrSeq RNA Exome Reference Gide 18 Transfer 15 µl spernatant to the corresponding well of the ALP plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Adenylate 3ʹ Ends A single 'A' ncleotide is added to the 3ʹ ends of the blnt fragments to prevent them from ligating to each other dring the adapter ligation reaction. A corresponding single 'T' ncleotide on the 3ʹ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensres a low rate of chimera (concatenated template) formation. Consmables ATL (A-Tailing Mix) RSB (Resspension Bffer) Microseal 'B' adhesive seals Bcket with ice Preparation 1 Prepare the following consmables. Item Storage Instrctions ATL -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Preheat 2 microheating systems, one to 37 C and another to 70 C. Procedre 1 Centrifge ATL at 600 g for 5 seconds. 2 Add 2.5 µl RSB to each well. 3 Add 12.5 µl ATL to each well. 4 Apply the seal and shake at 1800 rpm for 2 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on the 37 C microheating system with the lid closed for 30 mintes. 7 Move to the 70 C microheating system with the lid closed for 5 mintes. 8 Place on ice for 1 minte. Ligate Adapters This process ligates mltiple indexing adapters to the ends of the ds cdna fragments, which prepares them for hybridization onto a flow cell. Index adapters mst be ordered separately from the Library Prep components. For information on compatible index adapters, see Spporting Information on page 30. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 12

16 TrSeq RNA Exome Reference Gide Consmables LIG (Ligation Mix) RNA Adapters RSB (Resspension Bffer) AMPre XP beads STL (Stop Ligation Bffer) Freshly prepared 80% ethanol (EtOH) 96-well midi plate 96-well Hard-Shell 0.3 ml PCR plate Microseal 'B' adhesive seals Abot Reagents Do not remove LIG from storage ntil instrcted to do so in the procedre. Retrn LIG to storage immediately after se. Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions RNA Adapters -25 C to -15 C Thaw at room temperatre for 10 mintes. Retrn to storage after se. STL -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Preheat a microheating system to 30 C. 3 Label a new Hard-Shell PCR plate PCR with a marker. 4 Label a new midi plate CAP with a marker. Procedre Add Index Adapters 1 Centrifge the RNA Adapter tbes at 600 g for 5 seconds. 2 Remove LIG from -25 C to -15 C storage. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 13

17 TrSeq RNA Exome Reference Gide 3 Add the following reagents in the order listed to each well. RSB (2.5 µl) LIG (2.5 µl) RNA adapters (2.5 µl) 4 Apply the seal and shake at 1800 rpm for 2 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on the 30 C microheating system with the lid closed for 10 mintes, and then place on ice. 7 Centrifge STL at 600 g for 5 seconds. 8 Add 5 µl STL to each well. 9 Apply the seal and shake at 1800 rpm for 2 mintes. 10 Centrifge at 280 g for 1 minte. Clean Up Ligated Fragments 1 Perform steps 2 throgh 16 sing the Rond 1 volmes. 2 Add AMPre XP beads to each well. Rond 1 Rond 2 AMPre XP beads 42 µl 50 µl 3 Apply the seal and shake at 1800 rpm for 2 mintes. 4 Incbate at room temperatre for 5 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 5 mintes. 11 Remove from the magnetic stand. 12 Add RSB to each well. Rond 1 Rond 2 RSB 52.5 µl 22.5 µl 13 Apply the seal and shake at 1800 rpm for 2 mintes. 14 Incbate at room temperatre for 2 mintes. 15 Centrifge at 280 g for 1 minte. 16 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 14

18 TrSeq RNA Exome Reference Gide 17 Transfer 50 µl spernatant to the corresponding well of the CAP plate. 18 Repeat steps 2 throgh 16 with the new plate sing the Rond 2 volmes. 19 Transfer 20 µl spernatant to the corresponding well of the PCR plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Enrich DNA Fragments This process ses PCR to selectively enrich those DNA fragments that have adapter molecles on both ends and to amplify the amont of DNA in the library. PCR is performed with PPC (PCR Primer Cocktail) that anneals to the ends of the adapters. Minimize the nmber of PCR cycles to avoid skewing the representation of the library. NOTE Fragments with no adapters cannot hybridize to srface-bond primers in the flow cell. Fragments with an adapter on 1 end can hybridize to srface bond primers, bt cannot form clsters. Consmables PMM (PCR Master Mix) PPC (PCR Primer Cocktail) RSB (Resspension Bffer) AMPre XP beads Freshly prepared 80% ethanol (EtOH) 96-well Hard-Shell 0.3 ml PCR plate Microseal 'A' film Microseal 'B' adhesive seals NOTE Use Microseal 'A' when sealing the plate before placing it on the thermal cycler. Use Microseal 'B' for other steps that reqire a sealed plate. Abot Reagents Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions PPC -25 C to -15 C Thaw at room temperatre. Invert to mix, then centrifge at 600 g for 1 minte. Do not vortex. Retrn to storage after se. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 15

19 TrSeq RNA Exome Reference Gide Item Storage Instrctions PMM -25 C to -15 C Thaw on ice. Invert to mix, then centrifge at 600 g for 1 minte. Do not vortex. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Save the following PCR program on the thermal cycler: Choose the preheat lid option and set to 100 C 98 C for 30 seconds 15 cycles of: 98 C or 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 4 C Each well contains 50 µl 3 Label a new midi plate PPP with a marker. 4 Label a new Hard-Shell PCR plate TSP1 with a marker. Procedre Amplify DNA Fragments 1 Place on ice and add 5 μl PPC to each well. 2 Add 25 µl PMM to each well. 3 Apply the seal and shake at 1600 rpm for 2 mintes. 4 Apply the seal and centrifge at 280 g for 1 minte. NOTE If yo have separate pre-pcr and post-pcr areas, move to the post-pcr area. 5 Place on the preprogrammed thermal cycler and rn the PCR program. Clean Up Amplified DNA 1 Centrifge at 280 g for 1 minte. 2 Add 50 µl AMPre XP beads to each well. 3 Apply the seal and shake at 1800 rpm for 2 mintes. 4 Incbate at room temperatre for 15 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 7 Remove and discard all spernatant from each well. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 16

20 TrSeq RNA Exome Reference Gide 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 15 mintes. 11 Remove from the magnetic stand. 12 Add 17.5 µl RSB to each well. 13 Apply the seal and shake at 1800 rpm for 2 mintes. 14 Incbate at room temperatre for 2 mintes. 15 Centrifge at 280 g for 1 minte. 16 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 17 Transfer 15 µl spernatant to the corresponding well of the TSP1 plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Check Libraries Qantify Library Qantify yor library sing an Advanced Analytical Fragment Analyzer or Agilent Technologies 2100 Bioanalyzer. As an alternative, qantify sing PicoGreen. Check Library Qality 1 If sing a Standard Sensitivity NGS Fragment Analysis Kit on an Advanced Analytical Fragment Analyzer: a b Dilte the DNA library 1:1 with RSB. Rn 1 µl dilted DNA library. 2 If sing a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, rn 1 µl ndilted DNA library. 3 Check the size and prity of the sample. Expect the final prodct to be a band at ~ bp. Figre 7 Example Library Size Distribtion with Peak at ~300 bp (Before Normalization) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 17

21 TrSeq RNA Exome Reference Gide NOTE A ble dye is sed in the Assay reagents to aid in loading and collection. The dye appears as a characteristic peak at bp and is not indicative of isses with the final library. Figre 8 shows the distribtion of only the ble dye. Figre 8 Reagent Ble Dye Distribtion on BA DNA 1000 chip Hybridize Probes This step combines DNA libraries containing niqe indexes into a single pool, and then binds targeted regions of the DNA with captre probes. Consmables CT3 (Captre Target Bffer 3) CEX (Coding Exome Oligos) RSB (Resspension Bffer) 96-well Hard-Shell 0.3 ml PCR plate Microseal 'B' adhesive seal [Optional] Amicon Ultra-0.5 centrifgal filter nit (0.5 ml, 30 kda) (1 per pooled sample) Abot Reagents Before sing CT3, vortex to resspend the soltion. Make sre that no crystal strctres are present. If crystals and clodiness are observed, vortex ntil the soltion is clear. Preparation 1 Prepare the following consmables. Item Storage Instrctions CEX -25 C to -15 C Thaw at room temperatre. CT3-25 C to -15 C Thaw at room temperatre. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Save the RNA HYB program on the thermal cycler: Choose the preheat lid option and set to 100 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 18

22 TrSeq RNA Exome Reference Gide 95 C for 10 mintes 18 cycles of 1 minte each, starting at 94 C, then decreasing 2 C per cycle 58 C for 90 mintes NOTE Hybridizing longer than the programmed 2 hors reslts in a high degree of nonspecific binding. 3 Label a new Hard-Shell PCR plate REH1 (RNA Exome Hyb 1) with a marker. Pool Libraries Combine 200 ng of each DNA library for pooling. NOTE The TrSeq RNA Exome workflow contains enogh of each reagent for p to 1 to 4-plex pooling. For best reslts, pool only 4 or fewer libraries. Pooling an odd nmber of libraries does not affect data. For example, for 5 libraries, yo can pool 2 libraries and 3 libraries or 4 libraries and 1 library. Table 2 DNA Libraries for Enrichment Library Pool Complexity Total DNA Library Mass (ng) 1-plex plex plex plex 800 If the total volme is > 45 µl, se a vacm concentrator or Amicon Ultra-0.5 centrifgal filter nit (0.5 ml, 30 kda) to concentrate the pooled sample to 45 µl. If yo are sing a vacm concentrator, se a no heat setting and a medim drying rate. If yo are sing an Amicon Ultra-0.5 centrifgal filter nit (0.5 ml, 30 kda), it is not reqired to rinse the device before se. Most of the volme filters throgh in 5 mintes, bt p to 30 mintes can be reqired, depending on the starting volme. If the total volme is < 45 µl, increase the volme to 45 µl with RSB. Procedre 1 Add the following items in the order listed to each well of the REH1 plate. DNA library sample or pool (45 µl) CT3 (50 µl) CEX (5 µl) 2 For single-plex, add the following items in the order listed to each well of the REH1 plate: DNA library sample 200ng (11.25 µl) CT3 (12.5 µl) CEX (1.25 µl) 3 Shake at 1200 rpm for 1 minte. 4 Centrifge at 280 g for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 19

23 TrSeq RNA Exome Reference Gide 5 Place on the thermal cycler and rn the RNA HYB program. For 2-4 plex pools, each well contains 100 µl. For single-plex pools, each well contains 25 µl. 6 Remove from the thermal cycler immediately after the 90-minte incbation. Captre Hybridized Probes This step ses SMB (Streptavidin Magnetic Beads) to captre probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads. The enriched library is then elted from the beads and prepared for a second rond of hybridization. Consmables EE1 (Enrichment Eltion Bffer 1) ET2 (Elte Target Bffer 2) EWS (Enrichment Wash Soltion) HP3 (2 N NaOH) SMB (Streptavidin Magnetic Beads) 96-well Hard-Shell 0.3 ml PCR plate 96-well midi plate 1.7 ml microcentrifge tbe Microseal 'B' adhesive seals Abot Reagents EWS can be clody after reaching room temperatre. Vortex EWS before se. Make sre that yo se SMB (2 ml tbe) and not SPB (15 ml tbe) for this procedre. Invert and vortex SMB to mix before se. Discard eltion premix after se. Preparation 1 Prepare the following consmables. Item Storage Instrctions EE1-25 C to -15 C Thaw at room temperatre. Retrn to storage after se. EWS -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. HP3-25 C to -15 C Thaw at room temperatre. Retrn to storage after se. ET2 2 C to 8 C Let stand at room temperatre. Retrn to storage after se. SMB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Retrn to storage after se. 2 Preheat a microheating system with midi plate insert to 50 C. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 20

24 TrSeq RNA Exome Reference Gide 3 Label plates with a marker as follows. REW1 - midi REH2 - Hard-Shell PCR Procedre First Bind 1 Centrifge at 280 g for 1 minte. 2 Transfer all (~100 µl for pooled libraries, ~25 µl for 1-plex) to the corresponding well of the REW1 plate. NOTE If yo see a greater than 15% sample loss, do not proceed with the protocol. Poor sealing or insfficient heating of the lid can case sample loss. 3 Add 250 µl (62.5 µl for 1-plex) SMB to each well. 4 Shake at 1200 rpm for 5 mintes. 5 Incbate at room temperatre for 25 mintes. 6 Centrifge at 280 g for 1 minte. 7 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 8 Remove and discard all spernatant from each well. 9 Remove from the magnetic stand. First Wash 1 Wash 2 times as follows. a b c d e f g Add 200 µl (50 µl for 1-plex) EWS to each well. Shake at 1800 rpm for 4 mintes. Pipette to resspend the bead pellet frther. Place on the 50 C microheating system with the lid closed for 20 mintes. Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). Remove and discard all spernatant from each well. Remove from the magnetic stand. First Eltion 1 Create eltion premix in a 1.7 ml microcentrifge tbe, and then vortex to mix. EE1 (28.5 µl) HP3 (1.5 µl) 2 For 1-plex, create eltion premix in a 1.7 microcentrifge tbe, and then vortex to mix. EE1 (9.5 µl) HP3 (0.5 µl) 3 Add 23 µl (10 µl for 1-plex) eltion premix to each well. 4 Shake at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 2 mintes. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 21

25 TrSeq RNA Exome Reference Gide 6 Centrifge at 280 g for 1 minte. 7 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 8 Transfer 21 µl (9 µl for 1-plex) spernatant to the corresponding well of the REH2 plate. 9 Add 4 µl (1.7 µl for 1-plex) ET2 to each well. 10 Shake at 1200 rpm for 1 minte. 11 Centrifge at 280 g for 1 minte. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Perform Second Hybridization This step binds targeted regions of the enriched DNA with captre probes a second time. This second hybridization ensres high specificity of the captred regions. Consmables CT3 (Captre Target Bffer 3) CEX (Coding Exome Oligos) RSB (Resspension Bffer) Microseal 'B' adhesive seals Abot Reagents Before sing CT3, vortex to resspend the soltion. Make sre that no crystal strctres are present. If crystals and clodiness are observed, vortex ntil the soltion is clear. Preparation 1 Prepare the following consmables. Item Storage Instrctions CEX -25 C to -15 C Thaw at room temperatre. CT3-25 C to -15 C Thaw at room temperatre. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Procedre 1 Add the following reagents in the order listed to each well that contains a sample. RSB (20 µl) CT3 (50 µl) CEX (5 µl) 2 For 1-plex, add the following reagents in the order listed to each well that contains a sample. RSB (0.55 µl) CTE (12.5 µl) CEX (1.25 µl) 3 Shake at 1200 rpm for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 22

26 TrSeq RNA Exome Reference Gide 4 Centrifge at 280 g for 1 minte. 5 Place on the preprogrammed thermal cycler and rn the RNA HYB program. For 2-4 plex pools, each well contains 100 µl. For single-plex pools, each well contains 25 µl. 6 Remove from the thermal cycler immediately after the 90-minte incbation. Perform Second Captre This step ses SMB (Streptavidin Magnetic Beads) to captre probes hybridized to the targeted regions of interest. Two heated washes remove nonspecific binding from the beads. The enriched library is then elted from the beads and prepared for seqencing. Consmables EE1 (Enrichment Eltion Bffer 1) ET2 (Elte Target Bffer 2) EWS (Enrichment Wash Soltion) HP3 (2 N NaOH) SMB (Streptavidin Magnetic Beads) 96-well midi plates (2) 1.7 ml microcentrifge tbe Microseal 'B' adhesive seals Abot Reagents EWS can be clody after reaching room temperatre. Vortex EWS before se. Invert SMB to mix before se. Discard eltion premix after se. Preparation 1 Prepare the following consmables. Item Storage Instrctions EE1-25 C to -15 C Thaw at room temperatre. Retrn to storage after se. EWS -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. HP3-25 C to -15 C Thaw at room temperatre. Retrn to storage after se. ET2 2 C to 8 C Let stand at room temperatre. Retrn to storage after se. SMB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Retrn to storage after se. 2 Preheat a microheating system with midi plate insert to 50 C. 3 Label plates with a marker as follows. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 23

27 TrSeq RNA Exome Reference Gide REW1 - midi REH2 - Hard-Shell PCR Procedre Second Bind 1 Centrifge at 280 g for 1 minte. 2 Transfer all (~100 µl; ~25 µl for 1-plex) spernatant to the corresponding well of the REW2 plate. NOTE If yo see a greater than 15% sample loss, do not proceed with the protocol. Poor sealing or insfficient heating of the lid can case sample loss. 3 Add 250 µl (62.5 µl for 1-plex) SMB to each well. 4 Shake at 1200 rpm for 5 mintes. 5 Incbate at room temperatre for 25 mintes. 6 Centrifge at 280 g for 1 minte. 7 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 8 Remove and discard all spernatant from each well. 9 Remove from the magnetic stand. Second Wash 1 Wash 2 times as follows. a b c d e f g Add 200 µl (50 µl for 1-plex) EWS to each well. Shake at 1800 rpm for 4 mintes. Pipette to resspend the bead pellet frther. Place on the 50 C microheating system with the lid closed for 20 mintes. Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). Remove and discard all spernatant from each well. Remove from the magnetic stand. Second Eltion 1 Create eltion premix in a 1.7 ml microcentrifge tbe, and then vortex to mix. EE1 (28.5 µl) HP3 (1.5 µl) 2 For 1-plex, create eltion premix in a 1.7 ml microcentrifge tbe, and then vortex to mix. EE1 (9.5 µl) HP3 (0.5 µl) 3 Add 23 µl (10 µl for 1-plex) eltion premix to each well. 4 Shake at 1800 rpm for 2 mintes. 5 Incbate at room temperatre for 2 mintes. 6 Centrifge at 280 g for 1 minte. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 24

28 TrSeq RNA Exome Reference Gide 7 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 8 Transfer 21 µl (9 µl for 1-plex) spernatant to the corresponding well of the REC1 plate. 9 Add 4 µl (1 µl for 1-plex) ET2 to each well. 10 Shake at 1800 rpm for 1 minte. 11 Centrifge at 280 g for 1 minte. Clean Up Captred Library This step ses AMPre XP beads to prify the captred library before PCR amplification. Consmables RSB (Resspension Bffer) AMPre XP beads Freshly prepared 80% ethanol (EtOH) 96-well Hard-Shell 0.3 ml PCR plate Microseal 'B' adhesive seals Abot Reagents Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. 3 Label a new Hard-Shell PCR plate RAA with a marker. Procedre 1 Add 45 μl (18 μl for 1-plex) AMPre XP beads to each well. 2 Apply the seal and shake at 1800 rpm for 1 minte. 3 Incbate at room temperatre for 5 mintes. 4 Centrifge at 280 g for 1 minte. 5 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 6 Remove and discard all spernatant from each well. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 25

29 TrSeq RNA Exome Reference Gide 7 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 8 Use a 20 μl pipette to remove residal EtOH from each well. 9 Air-dry on the magnetic stand for 5 mintes. 10 Remove from the magnetic stand. 11 Add 27.5 μl RSB to each well. 12 Apply the seal and shake at 1800 rpm for 1 minte. 13 Incbate at room temperatre for 2 mintes. 14 Centrifge at 280 g for 1 minte. 15 Place on a magnetic stand and wait ntil the liqid is clear (~2 mintes). 16 Transfer 25 µl spernatant to the corresponding well of the REA plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Amplify Enriched Library This step ses a 10-cycle PCR program to amplify the enriched library. Consmables EPM (Enhanced PCR Mix) PPC (PCR Primer Cocktail) Microseal 'A' film Microseal 'B' adhesive seal NOTE Use Microseal 'A' when sealing the plate before placing on the thermal cycler. Use Microseal 'B' for other steps that reqire a sealed plate. Preparation 1 Prepare the following consmables. Item Storage Instrctions EPM -25 C to -15 C Thaw on ice. PPC -25 C to -15 C Thaw at room temperatre. 2 Save the following EPM AMP program on the thermal cycler: Choose the preheat lid option and set to 100 C 98 C for 30 seconds 10 cycles of: 98 C for 10 seconds Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 26

30 TrSeq RNA Exome Reference Gide 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 10 C Each well contains 50 µl Procedre 1 Add 5 µl PPC to each well. 2 Add 20 µl EPM to each well. The total volme per well is 50 µl. 3 Apply the seal and shake at 1200 rpm for 1 minte. 4 Centrifge at 280 g for 1 minte. 5 Place on the preprogrammed thermal cycler and rn the EPM AMP program. SAFE STOPPING POINT If yo are stopping, seal the plate and store at 2 C to 8 C for p to 2 days. Clean Up Amplified Enriched Library This step ses AMPre XP beads to prify the enriched library and remove nwanted prodcts. Consmables RSB (Resspension Bffer) AMPre XP beads Freshly prepared 80% ethanol (EtOH) 96-well Hard-Shell 0.3 ml PCR plate 96-well midi plate Microseal 'B' adhesive seals Abot Reagents Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. 3 Label a new Hard-Shell PCR plate REL with a marker. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 27

31 TrSeq RNA Exome Reference Gide 4 Label a new midi plate REC2 with a marker. Procedre 1 Centrifge at 280 g for 1 minte. 2 Transfer 50 µl to the corresponding well of the REC2 plate. 3 Add 90 µl AMPre XP beads to each well. 4 Apply the seal and shake RAC2 at 1800 rpm for 1 minte. 5 Incbate at room temperatre for 5 mintes. 6 Centrifge at 280 g for 1 minte. 7 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 8 Remove and discard all spernatant from each well. 9 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 10 Use a 20 μl pipette to remove residal EtOH from each well. 11 Air-dry on the magnetic stand for 5 mintes. 12 Remove from the magnetic stand. 13 Add 32 µl (8 µl for 1-plex) RSB to each well. 14 Apply the seal and shake at 1800 rpm for 1 minte. 15 Incbate at room temperatre for 2 mintes. 16 Centrifge at 280 g for 1 minte. 17 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 18 Transfer 30 µl (7.5 µl for 1-plex) spernatant to the corresponding well of the REL plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Check Enriched Libraries Perform the following procedres to check the qality of the enriched library. Qantify Libraries To achieve the highest-qality data on Illmina seqencing platforms, it is important to create optimm clster densities across every lane of the flow cell. Optimizing clster densities reqires accrate qantification of DNA libraries. 1 Qantify the libraries sing qpcr (preferred method) according to the Illmina Seqencing Library qpcr Qantification Gide (docment # ) or florometric method. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 28

32 TrSeq RNA Exome Reference Gide Assess Qality [Optional] 1 Load 1 µl 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 distribtion of DNA fragments with a size range from ~200 bp 1 kb. Follow manfactrer instrctions for either the Advanced Analytical Technologies Fragment Analyzer or Agilent Technologies 2100 Bioanalyzer, depending on the kit yo are sing. Depending on the level of indexing, insert size distribtion can vary slightly; however the sample peak mst not be significantly shifted compared to the example in Figre 9. Figre 9 Example TrSeq RNA Exome workflow Post-Enrichment Library Distribtion 3 Proceed to clster generation. For more information, see the system gide for yor Illmina platform. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 29

33 Appendix A Spporting Information Spporting Information Introdction 30 Prodct Contents 30 Consmables and Eqipment 32 Index Adapter Seqences 34 Acronyms 34 Introdction The protocol described in this gide assmes that yo have reviewed the contents of this section, confirmed workflow contents, and obtained all reqired consmables and eqipment. Prodct Contents Make sre that yo have all of the reagents identified in this section before starting the workflow. The following library prep, index adapter, enrichment, and coding exome oligo components are available to order throgh Illmina to spport the TrSeq RNA Exome workflow. Library Prep Component Catalog # TrSeq RNA Library Prep for Enrichment (48 samples) Enrichment Component Catalog # TrSeq RNA Enrichment (12 enrichments) Panel Component Catalog # Exome Panel (45 mb, 96 samples) Index Adapter Component Catalog # TrSeq RNA Single Indexes (12 indexes, 24 samples) Set A TrSeq RNA Single Indexes (12 indexes, 24 samples) Set B Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 30

34 TrSeq RNA Exome Reference Gide TrSeq RNA Library Prep for Enrichment (48 Samples) Library Prep for Enrichment, Store at -25 C to -15 C Qantity Reagent Description 1 FSA First Strand Synthesis Act D Mix 1 PMM PCR Master Mix 1 PPC PCR Primer Cocktail 1 SMM Second Strand Marking Master Mix 1 EPH Eltion Primer Fragmentation Mix 1 LIG Ligation Mix 1 ATL A-Tailing Mix 1 STL Stop Ligation Bffer 1 RSB Resspension Bffer TrSeq RNA Enrichment (12 Enrichments) This component is made p of two boxes with separate storage conditions. Sfficient reagents are provided to spport 12 enrichments of 1-plex p to 4-plex. Enrichment Reagents - Box 1, Store at 2 C to 8 C Qantity Reagent Description 1 ET2 Elte Target Bffer 2 3 SMB Streptavidin Magnetic Beads Enrichment Component - Box 2, Store at -25 C to -15 C Qantity Reagent Description 1 CT3 Captre Target Bffer 3 2 EE1 Enrichment Eltion Bffer 1 4 EPM Enhanced PCR Mix 2 EWS Enrichment Wash Soltion 1 HP3 2N NaOH 1 PPC PCR Primer Cocktail 1 RSB Resspension Bffer Coding Exome Oligos, Store at -25 C to -15 C Qantity Reagent Description 4 CEX Coding Exome Oligos Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 31