TruSeq RNA Exome Reference Guide Document # 1000000039582 v01 ILLUMINA PROPRIETARY March 2018 For Research Use Only. Not for use in diagnostic procedures. TruSeq RNA Exome Reference Guide 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. 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TruSeq RNA Exome Reference Guide Revision History Document Date Description of Change 1000000039582 v01 March • Changed Prepare for Pooling heading to Prepare Adapter Setup 2018 • Add Index Adapters procedure, removed the "Centrifuge STL at 600 x g for 5 seconds." step. • Enrich DNA Fragments|Consumables, added 96-well midi plate • Amplify DNA Fragment changed shake time to 20 seconds. • Hybridize Probes| Preparation, changed "58C for 90 minutes" to "58C for forever for 90 minutes." • Clean UP Amplified DNA, removed "Centrifuge at 280 x g for 1 minute.", added "Transfer contents from PCR plate to the CPP plate.", Incubate step 4 set to 5 minutes, Air-dry step set to 5 minutes. • Check Library Quality, corrected graphics. 1000000039582 v00 October Initial release. 2017 Document # 1000000039582 v01 iii For Research Use Only. Not for use in diagnostic procedures. Table of Contents Chapter 1 Overview 1 Introduction 1 RNA Input Recommendations 1 Additional Resources 4 Chapter 2 Protocol 6 Introduction 6 Tips and Techniques 6 Library Prep Workflow 8 Prepare Adapter Setup 9 Fragment RNA 9 Synthesize First Strand cDNA 10 Synthesize Second Strand cDNA 11 Adenylate 3ʹ Ends 13 Ligate Adapters 14 Enrich DNA Fragments 16 Check Libraries 18 First Hybridization of Probes 19 First Capture of Hybridized Probes 21 Second Hybridization of Probes 23 Second Capture of Hybridized Probes 24 Clean Up Captured Library 26 Amplify Enriched Library 28 Clean Up Amplified Enriched Library 29 Check Enriched Libraries 30 Appendix A Supporting Information 32 Introduction 32 Product Contents 32 Consumables and Equipment 34 Acronyms 36 Technical Assistance 38 Document # 1000000039582 v01 iv For Research Use Only. Not for use in diagnostic procedures. Chapter 1 Overview Introduction 1 RNA Input Recommendations 1 Additional Resources 4 Introduction This protocol explains how to convert total RNA into a library of known strand origin, and then capture the coding regions of the transcriptome using reagents for the Illumina® TruSeq® RNA Exome workflow. The library is suitable for subsequent cluster generation and DNA sequencing. The RNA is fragmented using divalent cations under elevated temperature. cDNA is generated from the cleaved RNA fragments using random priming during first and second strand synthesis. Then, sequencing adapters are ligated to the resulting double-stranded cDNA fragments. The coding regions of the transcriptome are captured from this library using sequence-specific probes to create the final library. This library prep protocol offers: u High data quality even from degraded or FFPE-derived RNA samples u Input requirement as low as 10 ng for fresh/frozen samples and 20 ng for FFPE samples u Uniform capture of the coding transcriptome, reducing sequencing requirement while maintaining discovery power u Up to 24 unique indexes and 4-plex pre-enrichment pooling for the most efficient use of your sequencing read budget u Single-plex enrichment for up to 48 samples u Strand information on RNA transcripts u High throughput automation-friendly procedures RNA Input Recommendations Total RNA Input u The protocol is optimized for 10–100 ng of human total RNA. u Lower amounts might result in low yield and inefficient ligation. u The protocol has been tested using 10 ng of high-quality universal human reference total RNA as input. u Use of RNA from other tissues or lower quality RNA might require further optimization to determine the input amount. Document # 1000000039582 v01 1 For Research Use Only. Not for use in diagnostic procedures. TruSeq RNA Exome Reference Guide u Determine the quality of the RNA starting material. The fragmentation conditions are optimized for high-quality RNA. u Use the Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity RNA Analysis Kit to determine the quality of your starting material. u The following figure shows a Universal Human Reference (UHR) starting RNA Bioanalyzer trace. Figure 1 Starting RNA Bioanalyzer Trace u Degraded or FFPE RNAs are shorter than full length RNA. u DNA contamination causes an underestimation of the amount of RNA used. Table 1 FFPE RNA Input Recommendations Quality DV200 Input Requirement Per Reaction High > 70% 20 ng Medium 50–70% 20–40 ng Low 30–50% 40–100 ng Too degraded < 30% Not recommended u For successful library prep, use an RNA isolation method that includes a reverse-crosslinking step and DNase1 treatment, such as the QIAGEN RNeasy FFPE Kit or QIAGEN AllPrep DNA/RNA FFPE Kit. u Use NanoDrop to determine FFPE RNA concentration. u For samples that border a quality classification, error towards the higher end of the input recommendation. NOTE For more information on calculating the DV200, see the Evaluating RNA Quality from FFPE Samples tech note for the TruSeq RNA Exome workflow. See Additional Resources on page 1 for information on how to download the tech note from the Illumina website. u If starting with FFPE RNA, the sample input amount is based on sample quality. Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality. Document # 1000000039582 v01 2 For Research Use Only. Not for use in diagnostic procedures. TruSeq RNA Exome Reference Guide u The following are examples of high, medium, and low quality FFPE traces, plus a trace of FFPE quality that is not recommended for use with TruSeq RNA Exome workflow. Figure 2 Example: High Quality FFPE (DV200 = 77%) Figure 3 Example: Medium Quality FFPE (DV200 = 55%) Figure 4 Example: Low Quality FFPE (DV200 = 30%) Document # 1000000039582 v01 3 For Research Use Only. Not for use in diagnostic procedures. TruSeq RNA Exome Reference Guide Figure 5 Example: FFPE Quality Not Recommended for Use (DV200 = 8%) Positive Control Use Agilent Technologies Human UHR total RNA (catalog # 740000) as a positive control sample for this protocol. Additional Resources The following documentation is available for download from the Illumina website. Resource Description Custom Protocol Selector A wizard for generating customized end-to-end documentation that is tailored to the library prep method, run parameters, and analysis method used for the sequencing run. TruSeq RNA Exome Checklist Provides a checklist of the protocol steps. The checklist is intended for experienced (document # 1000000040889) users. Evaluating RNA Quality from Provides effectivity profiles for FFPE RNA. FFPE Samples tech note TruSeq Library Prep Pooling Provides TruSeq pooling guidelines for preparing libraries for Illumina sequencing Guide (document # 15042173) systems that require balanced index combinations. Review this guide before beginning library preparation. Illumina Adapter Sequences Provides