RNA‐Seq: A Method for Comprehensive Transcriptome Analysis

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

A recently developed technique called RNA Sequencing (RNA?Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger sequencing? and microarray?based methods. In the RNA?Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next?generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole?genome transcriptome map. RNA?Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5? and 3? ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA?Seq using the Illumina sequencing platform. Curr. Protoc. Mol. Biol. 89:4.11.1?4.11.13. © 2010 by John Wiley & Sons, Inc.

Keywords: RNA?Seq; transcriptome; high?throughput sequencing; gene expression; annotation; cDNA library preparation

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Introduction
Basic Protocol 1: cDNA Library Preparation Using Fragmented Double‐Stranded cDNA
Alternate Protocol 1: cDNA Library Preparation Using Hydrolyzed or Fragmented RNA
Support Protocol 1: Purification of Fragmented RNA by Ethanol Precipitation
Support Protocol 2: Purification of cDNA Fragments
Support Protocol 3: DNA Sequencing and Data Analysis
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: cDNA Library Preparation Using Fragmented Double‐Stranded cDNA

Materials

Total RNA
500 ng/µl oligo(dT)_12‐18 primers (Invitrogen; store at −80°C)
10 mM dNTP mix (Invitrogen)
Nuclease‐free, sterile H₂ O
50 ng/µl random hexamer primers (Invitrogen; store at –80°C)
5× first‐strand buffer (Invitrogen)
100 mM dithiothreitol (DTT)
200 U/µl SuperScript II Reverse Transcriptase (Invitrogen)
5× second‐strand buffer (Invitrogen)
10 U/µl E. coli DNA ligase
10 U/µl E. coli DNA polymerase I
2 U/µl E. coli RNase H
5 U/µl T4 DNA polymerase (Promega)
0.5 M EDTA, pH 8.0 ( appendix 22 )
QIAquick PCR Purification Kit including Buffer EB (Qiagen)
DNase I buffer (New England Biolabs)
DNase I enzyme (New England Biolabs)
End‐It DNA End‐Repair Kit (Epicentre Biotechnologies) including:
- 10× End‐Repair Buffer
- End‐Repair Enzyme Mix
- 10 mM ATP
- 2.5 mM dNTP mix
Klenow buffer (NEB buffer 2; New England Biolabs)
Klenow fragment (3′ to 5′ exo^– ; New England Biolabs)
1 mM dATP (prepare from 100 mM dATP; New England Biolabs); store in 25‐µl single‐use aliquots at –20°C
QIAquick MinElute PCR Purification Kit including Buffer EB (Qiagen)
T4 DNA ligase buffer (Promega)
Illumina Genomic DNA Sequencing Kit including:
- Illumina Adapter Mix (part no. 100521)
- Illumina PCR primer 1.1 (part no. 100537)
- Illumina PCR primer 2.1 (part no. 100538)
3 U/µl T4 DNA ligase (Promega)
2× Phusion High Fidelity Master Mix (Finnzymes, cat. no. F‐531; http://www.finnzymes.us/)
1.5% to 2% agarose gel in TAE buffer (unit 2.5 )
Qiagen Gel Extraction Kit including Buffer EB

Heat block
Thermal cycler
PCR tubes
Horizontal agarose gel electrophoresis system (unit 2.5 )
Disposable scalpels
NanoDrop spectrophotometer (Thermo Scientific)

Additional reagents and equipment for preparation of poly(A)⁺ RNA (unit 4.5 ) and agarose gel electrophoresis ( protocol 4 )

Alternate Protocol 1: cDNA Library Preparation Using Hydrolyzed or Fragmented RNA

Poly(A)⁺ RNA prepared from total RNA (unit 4.5 )
10× RNA fragmentation buffer (Ambion)
Stop‐reaction buffer (0.2 M EDTA, pH 8.0)

Additional reagents and equipment for purification of fragmented cDNA by ethanol precipitation ( protocol 3 )

Support Protocol 1: Purification of Fragmented RNA by Ethanol Precipitation

Materials

Tube containing fragmented RNA ( protocol 2 , step 3)
3 M sodium acetate pH 5.2
100% nuclease‐free ethanol
70% nuclease‐free ethanol
Nuclease‐free water

Support Protocol 2: Purification of cDNA Fragments

Materials

cDNA library to be isolated
TAE buffer ( appendix 22 )
Disposable scalpels
Qiagen Gel Extraction Kit

Additional reagents and equipment for agarose gel electrophoresis (unit 2.5 )

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Figures

Figure 4.11.1 Flow chart of steps involved in RNA‐Seq method (step numbers refer to ).

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Literature Cited

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