TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide (15059581 v02)

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide (15059581 v02)

TruSeq Stranded mRNA Library Prep for NeoPrep

Protocol Guide

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

Prepare Samples for Loading

Set Up Run and Load Library Card

Unload Libraries

Validate Libraries

Normalize Libraries

Pool Libraries

Acronyms

Technical Assistance

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ILLUMINA PROPRIETARY

Document # 15059581 v02

March 2016

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).

FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN

MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND

DAMAGE TO OTHER PROPERTY.

ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

© 2016 Illumina, Inc. All rights reserved.

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Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect,

MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA,

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

Beckman Coulter, Inc.

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

Illumina, Inc. The purchase of this product from Illumina, 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 by the buyer

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Prepare Samples for Loading

Preparation

1 Save the following mRNA Denaturation program on a thermal cycler:

}

Choose the preheat lid option and set to 100°C

}

65°C for 5 minutes

}

25°C for 5 minutes

} 25°C hold

Procedure

1 Dilute 25–100 ng total RNA with nuclease-free ultrapure water to a final volume of

12.5 µl in each well of a new PCR plate. Pipette to mix. Do not vortex.

2 Vortex RPB2 to resuspend.

3 Add 12.5 µl RPB2 to each well. Pipette to mix.

4 Place on the thermal cycler and run the mRNA Denaturation program.

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide

3

Set Up Run and Load Library Card

Procedure

1 Vortex the reagent plate for 3 seconds.

2 Centrifuge at 600 × g for 5 seconds.

3 Select Prepare Libraries on the Library Prep Instrument Welcome screen.

4 Do the following and then select Next.

}

If running in BaseSpace mode, select a run.

}

If running in standalone mode, use the following options to select a protocol:

} Select Select by barcode, and then scan the reagent plate barcode or enter the reagent plate serial number.

}

Select Select by name, and then select TruSeq Stranded mRNA.

5 Configure the run. Select Next.

6 Review the run and sample information. Select Next.

7 Enter the consumable tracking information. Select Next.

8 Place the library card on the library card stage.

WARNING

To avoid instrument damage, make sure that the library card guide is not on the library card.

9 Close the library card compartment door. Select Verify Library Card.

10 Place the library card guide on the library card.

11 Load the entire contents of the oil vial into the library card using the oil funnel.

WARNING

Use the required pipette tips. Other tips are not supported and can result in reagents not dispensing properly and run failure.

The loading angle of the pipette depends on the item being dispensed. The angle is specified in each step of the control software loading guide and is depicted in these procedures.

12 Insert pipette tips to the bottom of the wells of the prepared sample plate. Pipette up and down 1 time to mix.

13 Transfer 25 µl of prepared samples 1–8.

14 Transfer 25 µl of prepared samples 9–16.

15 If you are preparing < 16 samples, add 25 µl RSB to empty sample wells.

16 Transfer 125 µl of the large reagents i–iv.

17 Transfer 125 µl of the large reagents v–vii.

18 Vortex DMB until well-dispersed.

19 Add 80 µl DMB to the large reagent well viii.

20 Transfer 15 µl of small reagents 1–4, and then 5–8.

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Document # 15059581 v02

21 For small reagents 9–12: a Use a clean 8-tube strip to pierce the foil on the reagent wells. Discard the 8-tube strip.

b Transfer 15 µl of each reagent.

22 For small reagents 13–16: a Use a clean 8-tube strip to pierce the foil on the reagent wells. Discard the 8-tube strip.

b Transfer 15 µl of each reagent.

23 Transfer 5 µl of small reagents a–d, and then e–h.

24 Transfer 3 µl of adapters A–H.

25 Transfer 3 µl of adapters I–P.

26 Remove the library card guide.

WARNING

To avoid instrument damage, make sure that the library card guide is removed from the library card.

27 Close the library card compartment door. Select Start Run.

28 When the run is complete, select Next.

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide

5

Unload Libraries

WARNING

The used library card contains hazardous materials. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and a laboratory coat. Handle the used library card as chemical waste. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more information, see the SDS for this kit at support.illumina.com/sds.html.

Procedure

1 Add 10 µl RSB to each well of a new PCR plate labeled 1–16.

2 Open the library card compartment door and place the library card guide on the library card.

3 Use a 200 µl pipette to transfer 20 µl from library card collection wells 1L–8L, and then 9L–16L to corresponding wells 1–16 of the plate. Pipette to mix.

4 Centrifuge briefly.

5 Transfer the entire volume from plate wells 1–8, and then 9–16 to the center indent in the membrane of the corresponding library separation tubes 1–16.

6 Let stand for 10 seconds while the oil is absorbed in the tubes.

7 Transfer the entire volume from library separation tubes 1–8, and then 9–16 to the corresponding wells 1–16 of a new PCR plate.

8 Remove the library card and library card guide from the library card stage.

9 Discard the library card in accordance with applicable standards.

10 Close the library card compartment door, and then select Home.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.

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Document # 15059581 v02

Validate Libraries

Procedure

1 Quantify the libraries using qPCR according to the Illumina Sequencing Library qPCR

Quantification Guide (document # 11322363).

2 If using a Standard Sensitivity NGS Fragment Analysis Kit on an Advanced

Analytical Fragment Analyzer: a Dilute the DNA library 1:1 with RSB.

b Run 1 µl diluted DNA library.

3 If using a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, run 1 µl undiluted DNA library.

4 Check the size and purity of the sample. Expect the final product to be a band at

~300 bp.

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide

7

Normalize Libraries

Procedure

1 Transfer 5 µl from each well of the library plate to the corresponding wells of a midi plate.

2 Normalize each library to 10 nM with Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20.

Pipette to mix.

3 Select from the following options:

}

For libraries that do not require pooling, the protocol stops here. Proceed to cluster generation.

} For libraries that require pooling, proceed to

Pool Libraries

.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.

8

Document # 15059581 v02

Pool Libraries

Procedure

1 Determine the number of samples to combine for each pool.

2 Transfer 5 µl of each library to be pooled from the library plate to a single well of a new PCR plate. Pipette to mix.

3 Proceed to cluster generation.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 2 months.

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide

9

Acronyms

Acronym

DMB

RPB2

RSB

Definition

Digital Microfluidics Beads

RNA Purification Beads 2

Resuspension Buffer

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Document # 15059581 v02

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 2 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

China

Denmark

Finland

France

Germany

Hong Kong

Ireland

Italy

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

400.635.9898

80882346

0800.918363

0800.911850

0800.180.8994

800960230

1.800.812949

800.874909

Region

Japan

Netherlands

New Zealand

Norway

Singapore

Spain

Sweden

Switzerland

Taiwan

United Kingdom

Other countries

Contact Number

0800.111.5011

0800.0223859

0800.451.650

800.16836

1.800.579.2745

900.812168

020790181

0800.563118

00806651752

0800.917.0041

+44.1799.534000

Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html

.

Product documentation—Available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature.

TruSeq Stranded mRNA Library Prep for NeoPrep Protocol Guide

Illumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

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