Digital Gene Expression by Tag Sequencing on the Illumina Genome Analyzer

Sorana Morrissy1, Yongjun Zhao1, Allen Delaney1, Jennifer Asano1, Noreen Dhalla1, Irene Li1, Helen McDonald1, Pawan Pandoh1, Anna‐Liisa Prabhu1, Angela Tam1, Martin Hirst1, Marco Marra1

1 BCCA Genome Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.11
DOI:  10.1002/0471142905.hg1111s65
Online Posting Date:  April, 2010
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Abstract

This unit provides a protocol for performing digital gene expression profiling on the Illumina Genome Analyzer sequencing platform. Tag sequencing (Tag‐seq) is an implementation of the LongSAGE protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression profiles. The ultra‐high‐throughput sequencing capability of the Illumina platform allows the cost‐effective generation of libraries containing an average of 20 million tags, a 200‐fold improvement over classical LongSAGE. Tag‐seq has less sequence composition bias, leading to a better representation of AT‐rich tag sequences, and allows a more accurate profiling of a subset of the transcriptome characterized by AT‐rich genes expressed at levels below the threshold of detection of LongSAGE (Morrissy et al., 2009). Curr. Protoc. Hum. Genet. 65:11.11.1‐11.11.36 © 2010 by John Wiley & Sons, Inc.

Keywords: gene expression; Tag‐seq; Illumina; RNA; cDNA; tag; PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: First‐ and Second‐Strand cDNA Synthesis for Tag‐seq Library Construction
  • Basic Protocol 2: Tag Generation
  • Basic Protocol 3: PCR and Fragment Isolation
  • Basic Protocol 4: Preparing the Library for Illumina Sequencing
  • Alternate Protocol 1: Amplified Tag‐seq Library Construction (Tag‐seqLite)
  • Basic Protocol 5: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: First‐ and Second‐Strand cDNA Synthesis for Tag‐seq Library Construction

  Materials
  • RNAseZap (Ambion)
  • DEPC‐treated H 2O ( appendix 2D)
  • Total RNA sample: typically isolated using TriZOL (Invitrogen), AllPrep Mini Kit (Qiagen), or RiboPure Kit (Ambion), and DNase I treated
  • Oligo(dT) magnetic beads (Invitrogen)
  • Lysis/Binding buffer (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • Wash Buffer B (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • 1× First Strand Buffer (Invitrogen)
  • 5× First‐Strand Buffer (Invitrogen)
  • 4 U/µl RNaseOUT (Invitrogen)
  • 0.1 M DTT (Invitrogen)
  • 5 M betaine (Sigma), prepared using nuclease‐free H 2O
  • 10 mM dNTP mix (10 mM each dNTP; Invitrogen)
  • 200 U/µl Superscript II Reverse Transcriptase (Invitrogen)
  • 5× Second‐Strand buffer (Invitrogen)
  • 10 U/µl E. coli DNA ligase (Invitrogen)
  • 10 U/µl E. coli DNA polymerase (Invitrogen)
  • 2 U/µl E. coli RNase H (Invitrogen)
  • Wash Buffer C (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • 0.5 M EDTA (Invitrogen)
  • Wash Buffer D (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • 10× Buffer 4 (New England Biolabs)
  • Textured nitrile gloves (Fisher)
  • Bench Coat (bench protection paper; Fisher)
  • RNase‐free 1.5‐ml non‐stick (siliconized) microcentrifuge tubes (Ambion)
  • Magnetic stand (Invitrogen, cat. no. R670‐01)
  • 50‐ml conical polypropylene tubes (BD Falcon)
  • Clay Adams Nutator Shaker (VWR Scientific)
  • Thermomixers, 1.5 ml (Eppendorf)

Basic Protocol 2: Tag Generation

  Materials
  • 100× bovine serum albumin (BSA; New England Biolabs)
  • 10× Buffer 4 (New England Biolabs)
  • 10 U/µl NlaIII restriction endonuclease (New England Biolabs)
  • cDNA‐containing magnetic beads ( protocol 1, step 49)
  • Wash Buffer C (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • Wash Buffer D (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • 10× ligase buffer (from I‐SAGE Long Kit; Invitrogen, cat. no. T5000‐03)
  • DEPC‐treated H 2O ( appendix 2D)
  • 10 µM GEX Adapter 1 (from Illumina Tag Profiling Sample Prep Kit, cat. no. FC‐102‐1005)
  • 5 U/µl T4 DNA ligase (Invitrogen)
  • DNA Away (Molecular BioProducts)
  • 32 mM (800×) S‐adenosylmethionine (SAM)
  • 10× (and 1×) Buffer 4 (New England Biolabs)
  • 2 U/µl MmeI restriction endonuclease (New England Biolabs)
  • 1 U/µl shrimp alkaline phosphatase (Invitrogen)
  • 2‐ml Phase‐Lock Gel (PLG) tube (heavy; Fisher)
  • Phenol/chloroform/isoamyl alcohol (PCI; Fisher)
  • 3 M sodium acetate, pH 5.5 (Ambion; also see appendix 2D)
  • 20 mg/ml mussel glycogen
  • 100% and ice‐cold 70% ethanol
  • 1.5 µM GEX Adapter 2 (from Illumina Tag Profiling Sample Prep Kit, cat. no. FC‐102‐1005)
  • Thermomixers, 1.5 ml (Eppendorf)
  • Textured nitrile gloves (Fisher)
  • RNase‐free 1.5‐ml non‐stick (siliconized) microcentrifuge tubes (Ambion)
  • 16°C water bath (Fisher Isotemp 3016)
  • Magnetic stand (Invitrogen, cat. no. R670‐01)

Basic Protocol 3: PCR and Fragment Isolation

  Materials
  • RNaseZap (Ambion)
  • Ultrapure Water (Invitrogen)
  • 5× HF buffer (Finnzymes)
  • DMSO (Invitrogen)
  • 10 mM dNTP mix (10 mM each dNTP; Invitrogen)
  • GEX PCR Primers 1 and 2 (from Illumina Tag Profiling Sample Prep Kit, cat. no. FC‐102‐1005)
  • 2 U/µl Phusion Hot Start DNA Polymerase (Finnzymes)
  • 40% acrylamide (19:1 acrylamide:bis; BioRad)
  • 50× TAE buffer ( appendix 2D)
  • 10% (w/v) ammonium persulfate (BioRad; prepare immediately before use)
  • TEMED (BioRad)
  • Micro‐90 cleaning solution (Cole‐Parmer)
  • 10× bromphenol blue/xylene cyanol loading dye (see recipe)
  • 25‐bp DNA ladder (20 ng/µl; Invitrogen) in loading dye (5:1)
  • SybrGreen (Cambrex Bio Science Inc.)
  • Elution buffer: 5 parts low‐TE buffer (10 mM Tris⋅Cl, pH 7.4 and 1 mM EDTA; see appendix 2D) plus 1 part 7.5 M ammonium acetate
  • 3 M sodium acetate, pH 5.5 (Ambion, or see appendix 2D)
  • 20 mg/ml mussel glycogen (Roche Scientific)
  • 70% and 100% ethanol (anhydrous ethyl alcohol; Commercial Alcohol Inc., http://www.comalc.com/)
  • EB buffer (from Qiagen PCR Purification Kit)
  • Textured nitrile gloves (Fisher)
  • RNase‐free 1.5‐ml, 0.5‐ml, and 2‐ml microcentrifuge tubes (Ambion)
  • 0.2‐ml thin‐walled, RNase‐free PCR tubes (Ambion)
  • Peltier Thermal Cycler (MJ Research)
  • Glass plates for PAGE gels (Owl Scientific)
  • Bags for gel pouring (Fisher Scientific)
  • Casting tray (Owl Scientific)
  • Colored tape (asssorted; VWR Scientific)
  • Combs (15 well, 1.5 mm; Owl Scientific, cat. no. P1‐15D
  • Spacers (1.5 mm; Owl Scientific, cat. no. P1‐SD)
  • Gel pouches (Owl Scientific, cat. no. GP2‐25)
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Penguin Owl Electrophoresis System (Owl Scientific)
  • Power supply (LVC2kW, 48VDCV; Tyco Electronics)
  • 18‐G needle
  • Typhoon Gel Scanner (GE Healthcare)
  • Dark Reader (UV transilluminator) (InterScience; http://www.interscience.com/)
  • Bench Coat (bench protection paper; Fisher)
  • Heating block
  • SpinX columns (0.22‐µm; Costar)

Basic Protocol 4: Preparing the Library for Illumina Sequencing

  Materials
  • Sample: PCR product (library; see protocol 3, step 43)
  • Agilent DNA 1000 Kit (Agilent)
  • EB buffer (from Qiagen PCR Purification Kit) supplemented with 0.1% (v/v) Tween‐20
  • Agilent 2100 Bioanalyzer (Agilent)
  • Chip Priming Station (Agilent)
  • IKA Vortex Mixer (Agilent)
  • Illumina Genome Analyzer (Illumina)
  • Cluster Station (Illumina)

Alternate Protocol 1: Amplified Tag‐seq Library Construction (Tag‐seqLite)

  • Total RNA sample: typically isolated using TriZOL (Invitrogen), AllPrep Mini Kit (Qiagen), or RiboPure Kit (Ambion) and DNase I treated
  • LITE1/LITE TS primer mix (20 µM each; Integrated DNA Technologies, http://www.idtdna.com/) including:
    • Biotin‐AAG CAG TGG TAA CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT‐TVN
    • Lite TS primer, 10 µM: AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG
  • Nuclease‐free H 2O (Ambion; also see recipe for DEPC‐treated H 2O in appendix 2D)
  • 20 mM DTT
  • SMARTScribe Reverse Transcriptase (Clontech)
  • TE buffer, pH 8.0 (Invitrogen; also see appendix 2D)
  • Advantage 2 PCR Kit (Clontech) including:
    • 10× Advantage 2 Buffer
    • 50× Advantage 2 Polymerase Mix
  • Buffer PB (Qiagen)
  • Buffer PE (Qiagen)
  • Buffer EB (Qiagen)
  • M280 Streptavidin beads (Invitrogen)
  • 2× and 1× B+W buffer (see recipe)
  • Thin‐walled, frosted‐lid, RNase‐free PCR tubes (Ambion)
  • Microcentrifuge adapters for the PCR tubes
  • Peltier Thermal Cycler (MJ Research)
  • QIAquick spin columns and collection tubes (Qiagen)
  • NanoDrop spectrophotometer ( appendix 3D)
  • Agilent DNA 7500 Kit (Agilent) including:
    • DNA 7500 Gel Matrix
    • DNA 7500 Maker
    • DNA 7500 Ladder
    • Agilent DNA 7500 Chips
    • Agilent Chip Priming Station
    • IKA Works Vortexer
    • Agilent Electrode Cleaner
  • Additional reagents and equipment for quantitating DNA using NanoDrop spectrophotometer ( appendix 3D)
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Figures

Videos

Literature Cited

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