RNA‐Seq: A Method for Comprehensive Transcriptome Analysis

Ugrappa Nagalakshmi1, Karl Waern1, Michael Snyder1

1 Molecular, Cellular, and Developmental Biology Department, Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.11
DOI:  10.1002/0471142727.mb0411s89
Online Posting Date:  January, 2010
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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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Table of Contents

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

  • Total RNA
  • 500 ng/µl oligo(dT) 12‐18 primers (Invitrogen; store at −80°C)
  • 10 mM dNTP mix (Invitrogen)
  • Nuclease‐free, sterile H 2O
  • 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

  • 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

  • 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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Literature Cited

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