Agilent SureSelect XT HS2 RNA User Manual

SureSelect XT HS2 RNA System

Strand-Specific RNA Library Preparation and Target Enrichment for the Illumina Platform

Protocol

Version A1, September 2020

SureSelect platform manufactured with Agilent SurePrint Technology

For Research Use Only. Not for use in diagnostic procedures.

Agilent Technologies

Notices

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Manual Part Number

G9989-90000

Edition

Version A1, September 2020

Printed in USA

Agilent Technologies, Inc. 5301 Stevens Creek Blvd Santa Clara, CA 95051 USA

Acknowledgment

Technical Support

For US and Canada

Call (800) 227-9770 (option 3,4,4) Or send an e-mail to:

ngs.support@agilent.com

For all other regions

Agilent’s world-wide Sales and Support Center contact details for your location can be obtained at

www.agilent.com/en/contact-us/page.

Warranty

The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control.

Technology Licenses

The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.

Restricted Rights Legend

U.S. Government Restricted Rights. Software and technical data rights granted to the federal government include only those rights customarily provided to end user customers. Agilent provides this customary commercial license in Software and technical data pursuant to FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS

252.227-7015 (Technical Data - Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Computer Software Documentation).

Notice to Purchaser

This product is provided under an agreement between Bio-Rad Laboratories and Agilent Technologies Inc., and the manufacture, use, sale or import of this product is subject to U.S. Pat. No. 6,627,424 and EP Pat. No.1 283 875 81, owned by Bio-Rad Laboratories, Inc. Purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in PCR (but not including real-time PCR) in the Research Field (including all Applied Research Fields, including but not limited to forensics, animal testing, and food testing) and in real-time PCR in the Diagnostics and Prognostics Fields. No rights are granted for use of this product for real-time PCR in the Research Field, including all Applied Research Fields (including but not limited to forensics, animal testing and food testing).

Limited Use Label License: This product and its use are the subject of one or more issued and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft exclusively licensed to New England Biolabs, Inc. and sublicensed to Agilent Technologies. The purchase of this product from Agilent Technologies, Inc., its affiliates, or its 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 in research conducted 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.

2 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Safety Notices

CAUTION

A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

WARNING

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 3

In this Guide...

This guide provides an optimized protocol for preparation of target- enriched Illumina paired- end multiplexed sequencing libraries using the SureSelect XT HS2 RNA system.

1 Before You Begin

This chapter contains information that you should read and understand before you start an experiment.

2 Preparation of Input RNA and Conversion to cDNA

This chapter describes the steps to prepare, qualify and fragment the RNA samples, then convert RNA to cDNA fragments.

3 Library Preparation

This chapter describes the steps to prepare dual- indexed, molecular- barcoded cDNA sequencing libraries for target enrichment.

4 Hybridization and Capture

This chapter describes the steps to hybridize and capture the prepared cDNA library using a SureSelect or ClearSeq probe capture library.

5 Post-Capture Sample Processing for Multiplexed Sequencing

This chapter describes the steps for post- capture amplification and guidelines for sequencing sample preparation.

6 Reference

This chapter contains reference information, including component kit contents and index sequences.

4 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

What’s New in Version A1

• Updates to index pair sequence tables (page 80 through

• Updates to downstream sequencing support information

• Updates to thawing conditions in Table 13 on page 30

page 87) including updates to P5 index platform

descriptions and correction of well position typographical errors

(see Table 38 on page 67 and Note on page 79)

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 5

6 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Content

1 Before You Begin 7

Overview of the Workflow 8

Procedural Notes 9

Safety Notes 9

Materials Required 10

Optional Materials 15

2 Preparation of Input RNA and Conversion to cDNA 17

Step 1A. Prepare and qualify FFPE RNA samples 19 Step 1B. Prepare and fragment intact RNA samples 22 Step 2. Synthesize first-strand cDNA 24 Step 3. Synthesize second-strand cDNA 25 Step 4. Purify cDNA using AMPure XP beads 26

3 Library Preparation 29

Step 1. Prepare the Ligation master mix 31 Step 2. Repair and dA-Tail the cDNA 3' ends 32 Step 3. Ligate the molecular-barcoded adaptor 34 Step 4. Purify the sample using AMPure XP beads 35 Step 5. Amplify the adaptor-ligated cDNA library 37 Step 6. Purify the amplified library with AMPure XP beads 40 Step 7. Assess quality and quantity 42

4 Hybridization and Capture 45

Step 1. Hybridize cDNA libraries to the probe 46 Step 2. Prepare streptavidin-coated magnetic beads 51 Step 3. Capture the hybridized DNA using streptavidin-coated beads 52

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 5

Contents

5 Post-Capture Sample Processing for Multiplexed Sequencing 55

Step 1. Amplify the captured libraries 56 Step 2. Purify the amplified captured libraries using AMPure XP beads 59 Step 3. Assess sequencing library DNA quantity and quality 61 Step 4. Pool samples for multiplexed sequencing 64 Step 5. Prepare sequencing samples 66 Step 6. Do the sequencing run and analyze the data 68 Sequence analysis resources 73

6 Reference 75

Kit Contents 76

SureSelect XT HS2 Index Primer Pair Information 79

Troubleshooting Guide 91

Quick Reference Protocol 94

6 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

SureSelect XT HS2 RNA System Protocol

1 Before You Begin

Overview of the Workflow 8 Procedural Notes 9 Safety Notes 9 Materials Required 10 Optional Materials 15

Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.

NOTE

Agilent guarantees performance and provides technical support for the SureSelect reagents required for this workflow only when used as directed in this Protocol.

Agilent Technologies

1 Before You Begin

Overview of the Workflow

The SureSelect XT HS2 RNA workflow for the preparation of NGS- ready libraries is summarized in Figure 1.

Figure 1 Overall target-enriched RNA sequencing sample preparation workflow.

8 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Procedural Notes

• To prevent contamination of reagents by nucleases, always wear

powder- free laboratory gloves and use dedicated solutions and pipettors with nuclease- free aerosol- resistant tips.

• Use best- practices to prevent PCR product and ribonuclease

contamination of samples throughout the workflow:

1 Assign separate pre- PCR and post- PCR work areas and use

dedicated equipment, supplies, and reagents in each area. In particular, never use materials designated to post- PCR work areas for pre- PCR segments of the workflow.

2 Maintain clean work areas. Clean the surfaces that pose the highest

risk of contamination daily using a 10% bleach solution, or equivalent.

3 Always use dedicated pre- PCR pipettors with nuclease- free

aerosol- resistant tips to pipette dedicated pre- PCR solutions.

4 Wear powder- free gloves. Use good laboratory hygiene, including

changing gloves after contact with any potentially- contaminated surfaces.

• For each protocol step that requires removal of tube cap strips, reseal

the tubes with a fresh strip of domed caps. Cap deformation may result from exposure of the cap strips to the heated lid of the thermal cycler and from other procedural steps. Reuse of strip caps can cause sample loss, sample contamination, or imprecision in sample temperatures during thermal cycler incubation steps.

• In general, follow Biosafety Level 1 (BSL1) safety rules.

• Possible stopping points, where samples may be stored at 4°C or –20°C,

are marked in the protocol. Do not subject the samples to multiple freeze/thaw cycles.

Before You Begin 1

Procedural Notes

Safety Notes

CAUTION

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 9

• Wear appropriate personal protective equipment (PPE) when working in the

laboratory.

1 Before You Begin

Materials Required

Materials required to complete the SureSelect XT HS2 RNA protocol are listed in the tables in this section. Select the preferred SureSelect XT HS2 RNA Reagent Kit format from Table 1, and a target enrichment probe from

Table 2. Then refer to Table 3 through Table 5 for additional materials

needed to complete the protocols using the selected kit format/RNA sample type.

Table 1 SureSelect XT HS2 RNA Reagent Kit Varieties

Description Kit Part Number

16 Reaction Kit

SureSelect XT HS2 RNA Reagent Kit G9989A (with Index Pairs 1–16) G9991A (with Index Pairs 1–96)

SureSelect XT HS2 RNA Reagent Kit with AMPure

XP/Streptavidin Beads

‡

G9990A (with Index Pairs 1–16) G9992A (with Index Pairs 1–96)

96 Reaction Kit

G9991B (with Index Pairs 97–192) G9991C (with Index Pairs 193–288) G9991D (with Index Pairs 289–384)

G9992B (with Index Pairs 97–192) G9992C (with Index Pairs 193–288) G9992D (with Index Pairs 289–384)

†

* 16-reaction kits contain enough reagents for 2 runs containing 8 samples per run.

† 96-reaction kits contain enough reagents for 4 runs containing 24 samples per run.

‡ AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.

10 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Before You Begin 1

Materials Required

Table 2 Compatible Probes

Probe Capture Library Design Target 16 Reactions 96 Reactions

Pre-designed Probes

SSel XT HS and XT Low Input Human All Exon V7 Genome 5191-4028 5191-4029

SureSelect XT Clinical Research Exome V2 Genome 5190-9491 5190-9492

SureSelect XT Mouse All Exon Genome 5190-4641 5190-4642

ClearSeq Comprehensive Cancer XT Genome 5190-8011 5190-8012

ClearSeq Inherited Disease XT Genome 5190-7518 5190-7519

ClearSeq RNA Kinome Transcriptome 5190-4801 5190-4802

Custom Probes

SureSelect Custom Tier1 1–499 kb

SureSelect Custom Tier2 0.5 –2.9 Mb

SureSelect Custom Tier3 3 –5.9 Mb

SureSelect Custom Tier4 6 –11.9 Mb

SureSelect Custom Tier5 12–24 Mb

Pre-designed Probes customized with additional Plus custom content

SSel XT HS and XT Low Input Human All Exon V7 Plus 1 Genome

SSel XT HS and XT Low Input Human All Exon V7 Plus 2 Genome

SureSelect XT Clinical Research Exome V2 Plus 1 Genome

SureSelect XT Clinical Research Exome V2 Plus 2 Genome

ClearSeq Comprehensive Cancer Plus XT Genome

ClearSeq Inherited Disease Plus XT Genome

Custom probes may designed for either genomic or transcriptomic targets. Please contact the SureSelect support team (see page 2) or your local representative for assistance with custom probe design and ordering for RNA library target enrichment.

Please visit the SureDesign

website to design the customized

Plus content and obtain ordering information. Contact the SureSelect support team (see

page 2) or your local

representative if you need assistance.

* Custom Probes designed August 2020 or later are produced using an updated manufacturing process; design-size Tier is

shown on labeling for these products. Custom Probes designed and ordered prior to August 2020 may be reordered, with these probes produced using the legacy manufacturing process; design-size Tier is not shown on labeling for the legacy-process products. Custom Probes of both categories use the same optimized target enrichment protocols detailed in this publication.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 11

1 Before You Begin

Materials Required

Table 3 Required Reagents

Description Vendor and Part Number Notes

1X Low TE Buffer Thermo Fisher Scientific p/n

12090-015, or equivalent

100% Ethanol (Ethyl Alcohol, 200 proof) Millipore p/n EX0276 —

Nuclease-free Water Thermo Fisher Scientific p/n

AM9930

AMPure® XP Kit 5 ml 60 ml 450 ml

Dynabeads MyOne Streptavidin T1 2 ml 10 ml 50 ml

QPCR Human Reference Total RNA Agilent p/n 750500 Control input RNA (optional)

Beckman Coulter Genomics

p/n A63880 p/n A63881 p/n A63882

Thermo Fisher Scientific p/n 65601 p/n 65602 p/n 65604D

10 mM Tris-HCl, pH 7.5-8.0, 0.1 mM EDTA

Water should not be DEPC-treated

Separate purchase not required for use

with SureSelect XT HS2 RNA Reagent Kits that include SureSelect DNA

AMPure Streptavidin Beads (Agilent p/n G9990A, G9992A, G9992B, G9992C, or G9992D)

XP Beads and SureSelect

12 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Before You Begin 1

Materials Required

CAUTION

Sample volumes exceed 0.2 ml in certain steps of this protocol. Make sure that the plasticware

used with the selected thermal cycler holds 0.25 ml per well.

Table 4 Required Equipment

Description Vendor and Part Number

Thermal Cycler with 96-well, 0.2 ml block Various suppliers

Plasticware compatible with the selected thermal cycler:

96-well plates or 8-well strip tubes Tube cap strips, domed

Low-adhesion tubes (RNase, DNase, and DNA-free)

1.5 mL

0.5 mL

Microcentrifuge Eppendorf microcentrifuge, model 5417C or equivalent

Plate or strip tube centrifuge Labnet International MPS1000 Mini Plate Spinner, p/n

96-well plate mixer Eppendorf ThermoMixer C, p/n 5382000023 and Eppendorf

Small-volume spectrophotometer NanoDrop 2000, Thermo Fisher Scientific p/n ND-2000 or

Multichannel pipette Rainin Pipet-Lite Multi Pipette or equivalent

Single channel pipettes (10-, 20-, 200-, and 1000-µl capacity) Rainin Pipet-Lite Pipettes or equivalent

Sterile, nuclease-free aerosol barrier pipette tips general laboratory supplier

Vortex mixer general laboratory supplier

Ice bucket general laboratory supplier

Powder-free gloves general laboratory supplier

Magnetic separator Thermo Fisher Scientific p/n 12331D or equivalent

Consult the thermal cycler manufacturer’s

recommendations

USA Scientific p/n 1415-2600 p/n 1405-2600

C1000 (requires adapter, p/n C1000-ADAPT, for use with strip tubes) or equivalent

SmartBlock 96 PCR, p/n 5306000006, or equivalent

equivalent

* Select a magnetic separator configured to collect magnetic particles on one side of each well. Do not use a magnetic sep-

arator configured to collect the particles in a ring formation.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 13

1 Before You Begin

Materials Required

Table 5 Nucleic Acid Analysis Platform Options--Select One

Description Vendor and Part Number

Agilent 4200/4150 TapeStation

Consumables:

96-well sample plates

96-well plate foil seals

8-well tube strips

8-well tube strip caps

RNA ScreenTape

RNA ScreenTape Sample Buffer

RNA ScreenTape Ladder

High Sensitivity RNA ScreenTape

High Sensitivity RNA ScreenTape Sample Buffer

High Sensitivity RNA ScreenTape Ladder

D1000 ScreenTape

D1000 Reagents

High Sensitivity D1000 ScreenTape

High Sensitivity D1000 Reagents

Agilent 2100 Bioanalyzer Instrument

Agilent 2100 Expert SW Laptop Bundle (optional)

Consumables:

RNA 6000 Pico Kit

RNA 6000 Nano Kit

DNA 1000 Kit

High Sensitivity DNA Kit

Agilent 5200/5300/5400 Fragment Analyzer Instrument

Consumables:

RNA Kit (15NT)

HS RNA Kit (15NT)

NGS Fragment Kit (1-6000 bp)

HS NGS Fragment Kit (1-6000 bp)

Agilent p/n G2991AA/G2992AA

Agilent p/n 5042-8502

Agilent p/n 5067-5154

Agilent p/n 401428

Agilent p/n 401425

Agilent p/n 5067-5576

Agilent p/n 5067-5577

Agilent p/n 5067-5578

Agilent p/n 5067-5579

Agilent p/n 5067-5580

Agilent p/n 5067-5581

Agilent p/n 5067-5582

Agilent p/n 5067-5583

Agilent p/n 5067-5584

Agilent p/n 5067-5585

Agilent p/n G2939BA

Agilent p/n G2953CA

Agilent p/n 5067-1513

Agilent p/n 5067-1511

Agilent p/n 5067-1504

Agilent p/n 5067-4626

Agilent p/n M5310AA/M5311AA/M5312AA

p/n DNF-471-0500

p/n DNF-472-0500

p/n DNF-473-0500

p/n DNF-474-0500

14 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Before You Begin 1

Optional Materials

Table 6 Supplier Information for Optional Materials

Description Vendor and Part Number Purpose

Tween 20 Sigma-Aldrich p/n P9416-50ML Sequencing library

storage (see page 69)

Optical Caps, 8× strip (flat) Consult the thermal cycler

manufacturer’s recommendations

MicroAmp Clear Adhesive Film Thermo Fisher Scientific p/n 4311971 Improved sealing for

PlateLoc Thermal Microplate Sealer with Small Hotplate and Peelable Aluminum Seal for PlateLoc Sealer

* Flat strip caps may be used instead of domed strip caps for protocol steps performed outside of the thermal cycler. Adhesive

film may be applied over the flat strip caps for improved sealing properties.

Please contact the SureSelect support team (see page 2) or your local representative for ordering information

Sealing wells for protocol steps performed outside of the thermal cycler

flat strip caps*

Sealing wells for protocol steps performed inside or outside of the thermal cycler

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 15

1 Before You Begin

Optional Materials

16 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

SureSelect XT HS2 RNA System Protocol

2 Preparation of Input RNA and Conversion to cDNA

This chapter describes the steps to prepare input RNA samples, including RNA fragmentation when required, and the steps to convert the RNA fragments to strand- specific cDNA prior to sequencing library preparation and target enrichment.

The protocol is compatible with both intact RNA prepared from fresh or fresh frozen samples and lower- quality RNA prepared from FFPE samples. For FFPE- derived RNA samples, begin the protocol using “Step 1A.

Prepare and qualify FFPE RNA samples” on page 19. For intact RNA

samples, begin the protocol using “Step 1B. Prepare and fragment intact

RNA samples” on page 22.

RNA sequencing library preparation requires RNA fragments sized appropriately for the NGS workflow. In this section of the protocol, intact total RNA samples are chemically- fragmented by treatment with metal ions present in the 2X Priming Buffer at elevated temperature. FFPE- derived RNA samples are already sufficiently fragmented. The FFPE samples must be combined with the same 2X Priming Buffer, but the mixtures are held on ice, preventing further fragmentation of the FFPE- derived RNA.

Protocols in this section for both intact RNA and FFPE sample types are applicable to either 2 x 100 bp or 2 x 150 bp read- length sequencing.

Agilent Technologies

2 Preparation of Input RNA and Conversion to cDNA

The protocol steps in this section use the components listed in Table 7. Thaw and mix each component as directed in Table 7 before use (refer to the Where Used column). Remove the AMPure XP beads from cold storage and equilibrate to room temperature for at least 30 minutes in preparation for use on page 26. Do not freeze the beads at any time.

Table 7 Reagents thawed before use in protocol

Kit Component Storage Location Thawing

Conditions

2X Priming Buffer (tube with purple cap)

First Strand Master Mix (amber tube with amber cap)

Second Strand Enzyme Mix (tube with blue cap or bottle)

Second Strand Oligo Mix (tube with yellow cap)

* The First Strand Master Mix contains actinomycin-D and is provided ready-to-use. Keep the reagent in the supplied amber

vial to protect the contents from exposure to light.

SureSelect cDNA Module (Pre PCR), –20°C

Thaw on ice then keep on ice

Thaw on ice for 30 minutes then keep on ice

Thaw on ice then keep on ice

Mixing Method

Vortexing page 21 (FFPE RNA) OR

Vortexing page 24

Vortexing page 25

Where Used

page 23 (intact RNA)

18 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Preparation of Input RNA and Conversion to cDNA 2

Step 1A. Prepare and qualify FFPE RNA samples

The instructions in this section are for FFPE- derived RNA samples. For intact (non- FFPE) RNA samples, instead follow the instructions in “Step

1B. Prepare and fragment intact RNA samples” on page 22.

Prepare total RNA from each FFPE sample in the run. The library preparation protocol requires 10–200 ng of FFPE total RNA in a 10 µl volume of nuclease- free water.

Consider preparing an additional sequencing library in parallel, using a high- quality control RNA sample, such as Agilent’s QPCR Human Reference Total RNA (p/n 750500). Use of this control is especially recommended during the first run of the protocol, to verify that all protocol steps are being successfully performed. Routine use of this control is helpful for any required troubleshooting, in order to differentiate any performance issues related to RNA input from other factors.

Before you begin the library preparation protocol, assess the initial quality of each sample in order to determine the appropriate reaction conditions at several steps in the workflow. Use the steps below to qualify each FFPE total RNA sample.

1 Use a small- volume spectrophotometer to determine sample absorbance

at 260 nm, 280 nm, and 230 nm. Determine the RNA concentration and the 260/280 and 260/230 absorbance ratio values for the sample.

High- quality RNA samples are indicated by values of approximately 1.8 to 2.0 for both ratios. Ratios with significant deviation from 2.0 indicate the presence of organic or inorganic contaminants, which may require further purification or may indicate that the sample is not suitable for use in RNA target enrichment applications.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 19

2 Preparation of Input RNA and Conversion to cDNA

Step 1A. Prepare and qualify FFPE RNA samples

2 Examine the starting size distribution of RNA in the sample using one

of the RNA qualification systems described in Table 8. Select the specific assay appropriate for your sample based on the RNA concentration determined in step 1 on page 19.

Determine the DV200 (percentage of RNA in the sample that is >200 nt) using the analysis mode described in Table 8. RNA molecules must be >200 nt for efficient conversion to cDNA library.

Table 8 RNA qualification platforms

Analysis Instrument RNA Qualification Assay Analysis to Perform

4200/4150 TapeStation RNA ScreenTape or High Sensitivity

RNA ScreenTape

2100 Bioanalyzer RNA 6000 Pico Chip or NanoChip Smear/Region analysis using 2100 Expert Software

5200 Fragment Analyzer RNA Kit (15NT) or HS RNA Kit

(15NT)

Region analysis using TapeStation Analysis Software

Analysis using ProSize Data Analysis Software

NOTE

Grading of FFPE RNA quality by RNA Integrity Number (RIN) is not recommended for this application.

3 Grade each RNA sample based on the percentage of RNA in the sample

>200 nucleotides, according to Table 9.

Table 9 Classification of FFPE RNA samples based on starting RNA size

Grade DV200 Recommended input

amount

Good FFPE RNA >50% 200 ng 10 ng

Poor FFPE RNA 20% to 50% 200 ng 50 ng

Inapplicable FFPE RNA <20% Not recommended for further processing

* For optimal results, prepare libraries from poor-grade FFPE RNA samples using a minimum of 50 ng

input RNA. Libraries may be prepared from 10–50 ng poor-grade FFPE RNA with potential negative impacts on yield or NGS performance.

Minimum input amount

4 Place 10 µl of each sample, containing 10–200 ng of FFPE total RNA in

nuclease- free water, into wells of a thermal cycler- compatible strip tube or PCR plate.

Poor- quality FFPE samples should contain at least 50 ng RNA.

20 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Preparation of Input RNA and Conversion to cDNA 2

Step 1A. Prepare and qualify FFPE RNA samples

5 Add 10 µl of 2X Priming Buffer to each sample well.

6 Mix well by pipetting up and down 15–20 times or seal the wells and

vortex at high speed for 5–10 seconds. Spin briefly to collect the liquid then place the RNA samples on ice.

NOTE

All samples, including highly degraded FFPE samples, must be combined with 2X Priming Buffer, which supplies the random primers for cDNA synthesis. FFPE RNA samples are not subjected to the high-temperature incubation step used for fragmentation in this buffer.

7 Proceed immediately to “Step 2. Synthesize first- strand cDNA” on

page 24.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 21

2 Preparation of Input RNA and Conversion to cDNA

Step 1B. Prepare and fragment intact RNA samples

The instructions in this section are for intact RNA prepared from fresh or fresh frozen samples. For FFPE- derived RNA samples, instead follow the instructions in “Step 1A. Prepare and qualify FFPE RNA samples” on page 19.

The 2X Priming Buffer used in this step includes both fragmentation agents and primers used for cDNA synthesis in the following steps. The fragmentation conditions shown in this section are appropriate for both 2 x 100 bp and 2 x 150 bp NGS read- length workflows.

1 Prepare total RNA from each sample in the run. The library preparation

protocol requires 10–200 ng of intact total RNA in a 10 µl volume of nuclease- free water.

Verify the RNA concentration and quality using a small volume spectrophotometer and one of the RNA qualification platforms listed in

Table 5 on page 14.

2 Preprogram a thermal cycler with the program in Table 10. Immediately

pause the program, and keep paused until samples are loaded in step 6.

Table 10 Thermal cycler program for fragmentation of intact RNA samples

Step Temperature Time

Step 1 94°C 4 minutes

Step 2 4°C 1 minute

Step 3 4°C Hold

* Use a reaction volume setting of 20 l, if required for thermal cycler set up.

NOTE

22 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

When using the SureCycler 8800 thermal cycler, the heated lid may be left on (default setting) throughout the RNA library preparation incubation steps. The heated lid must be on during the amplification and hybridization steps on page 38, page 47 and page 57.

Preparation of Input RNA and Conversion to cDNA 2

Step 1B. Prepare and fragment intact RNA samples

3 Place 10 µl of each sample, containing 10–200 ng total RNA in

nuclease- free water, into wells of a thermal cycler- compatible strip tube or PCR plate.

4 Add 10 µl of 2X Priming Buffer to each sample well.

5 Mix well by pipetting up and down 15–20 times or seal the wells and

vortex at high speed for 5–10 seconds. Spin briefly to collect the liquid.

6 Place the samples in the thermal cycler, and resume the thermal cycling

program in Table 10 for RNA fragmentation.

7 Once the thermal cycler program in Table 10 reaches the 4°C Hold

step, transfer the fragmented RNA sample plate or strip tube from the thermal cycler to ice or a cold block. Proceed immediately to “Step 2.

Synthesize first- strand cDNA” on page 24.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 23

2 Preparation of Input RNA and Conversion to cDNA

Step 2. Synthesize first-strand cDNA

CAUTION

The First Strand Master Mix used in this step is viscous. Mix thoroughly by vortexing at high speed for 5 seconds before removing an aliquot for use and after combining with other solutions. Pipetting up and down is not sufficient to mix this reagent.

The First Strand Master Mix is provided with actinomycin-D already supplied in the mixture. Do not supplement with additional actinomycin-D.

1 Preprogram a thermal cycler with the program in Table 11. Immediately

pause the program, and keep paused until samples are loaded in step 5.

Table 11 Thermal cycler program for first-strand cDNA synthesis

Step Temperature Time

Step 1 25°C 10 minutes

Step 2 37°C 40 minutes

Step 3 4°C Hold

* Use a reaction volume setting of 28 l, if required for thermal cycler set up.

2 Vortex the thawed vial of First Strand Master Mix for 5 seconds at high

speed to ensure homogeneity.

3 Add 8.5 µl of First Strand Master Mix to each RNA sample well.

4 Mix well by pipetting up and down 15–20 times or seal the wells and

vortex at high speed for 5–10 seconds. Spin briefly to collect the liquid.

5 Place the samples in the thermal cycler, and resume the program in

Table 11.

24 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Preparation of Input RNA and Conversion to cDNA 2

Step 3. Synthesize second-strand cDNA

CAUTION

The Second Strand Enzyme Mix used in this step is viscous. Mix thoroughly by vortexing at high speed for 5 seconds before removing an aliquot for use and after combining with other solutions. Pipetting up and down is not sufficient to mix this reagent.

1 Once the thermal cycler program in Table 11 begins the 4°C hold step,

transfer the samples to ice.

2 Preprogram the thermal cycler with the program in Table 12.

Immediately pause the program, and keep paused until samples are loaded in step 7

Table 12 Thermal cycler program for second-strand synthesis

Step Temperature Time

Step 1 16°C 60 minutes

Step 2 4°C Hold

* Use a reaction volume setting of 58 l, if required for thermal cycler set up.

3 Vortex the thawed vials of Second Strand Enzyme Mix and of Second

Strand Oligo Mix at high speed for 5 seconds to ensure homogeneity.

4 Add 25 µl of Second Strand Enzyme Mix to each sample well. Keep on

ice.

5 Add 5 µl of Second Strand Oligo Mix to each sample well, for a total

reaction volume of 58.5 µl. Keep on ice.

6 Mix well by pipetting up and down 15–20 times or seal the wells and

vortex at high speed for 5–10 seconds. Spin briefly to collect the liquid.

7 Place the plate or strip tubes in the thermal cycler, and resume the

program in Table 12.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 25

2 Preparation of Input RNA and Conversion to cDNA

Step 4. Purify cDNA using AMPure XP beads

1 Verify that the AMPure XP beads have been held at room temperature

for at least 30 minutes before use.

2 Prepare 400 µl of 70% ethanol per sample, plus excess, for use in

step 9.

NOTE

The freshly-prepared 70% ethanol may be used for subsequent purification steps run on the same day. The complete RNA Library Preparation protocol requires 1.2 mL of fresh 70% ethanol per sample and the Target Enrichment protocol requires an additional 0.4 mL of fresh 70% ethanol per sample.

3 Mix the bead suspension well so that the suspension appears

homogeneous and consistent in color.

4 Transfer the samples in the PCR plate or strip tube to room

temperature, then add 105 µl of the homogeneous bead suspension to each cDNA sample well.

5 Pipette up and down 15–20 times or cap the wells and vortex at high

speed for 5–10 seconds to mix. If the beads have splashed into the well caps, spin briefly to collect the samples, being careful not to pellet the beads.

6 Incubate samples for 5 minutes at room temperature.

7 Put the plate or strip tube into a magnetic separation device. Wait for

the solution to clear (approximately 2 to 5 minutes).

8 Keep the plate or strip tube in the magnetic stand. Carefully remove

and discard the cleared solution from each well. Do not touch the beads while removing the solution.

9 Continue to keep the plate or strip tube in the magnetic stand while

you dispense 200 µl of fresh 70% ethanol in each sample well.

10 Wait for 1 minute to allow any disturbed beads to settle, then remove

the ethanol.

11 Repeat step 9 and step 10 once for a total of two washes.

12 Seal the wells with strip caps, then briefly spin the samples to collect

the residual ethanol. Return the plate or strip tube to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette.

26 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

Preparation of Input RNA and Conversion to cDNA 2

Step 4. Purify cDNA using AMPure XP beads

13 Dry the samples by placing the unsealed plate or strip tube on the

thermal cycler, set to hold samples at 37°C, until the residual ethanol has just evaporated (up to 2 minutes).

NOTE

Stopping Point If you do not continue to the next step, seal the wells and store at 4°C

Do not dry the bead pellet to the point that the pellet appears cracked during any of the bead drying steps in the protocol. Elution efficiency is significantly decreased when the bead pellet is excessively dried.

14 Add 52 µl nuclease- free water to each sample well.

15 Seal the wells with strip caps, then vortex the plate or strip tube for

5 seconds. Verify that all beads have been resuspended, with no visible clumps in the suspension or bead pellets retained on the sides of the wells. Briefly spin to collect the liquid, being careful not to pellet the beads.

16 Incubate for 2 minutes at room temperature.

17 Put the plate or strip tube in the magnetic stand and leave until the

solution is clear (up to 5 minutes).

18 Remove 50 µl of cleared supernatant to a fresh PCR plate or strip tube

sample well and keep on ice. You can discard the beads at this time.

overnight or at –20°C for prolonged storage.

SureSelect XT HS2 RNA Library Preparation and Target Enrichment 27

2 Preparation of Input RNA and Conversion to cDNA

Step 4. Purify cDNA using AMPure XP beads

28 SureSelect XT HS2 RNA Library Preparation and Target Enrichment

+ 69 hidden pages

Agilent SureSelect XT HS2 RNA User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Overview of the Workflow

Procedural Notes

Safety Notes

Materials Required

Optional Materials

Step 1A. Prepare and qualify FFPE RNA samples

Step 1B. Prepare and fragment intact RNA samples

Step 2. Synthesize first-strand cDNA

Step 3. Synthesize second-strand cDNA

Step 4. Purify cDNA using AMPure XP beads