Paired‐End diTagging for Transcriptome and Genome Analysis

Patrick Ng1, Chia‐Lin Wei1, Yijun Ruan1

1 Genome Institute of Singapore
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.12
DOI:  10.1002/0471142727.mb2112s79
Online Posting Date:  July, 2007
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Abstract

The Paired‐End diTagging (PET) procedure enables one to obtain sequence information from both termini of any contiguous DNA fragment. This is achieved by a series of enzymatic manipulations that introduce MmeI sites directly flanking each DNA insert during the construction of a plasmid library. Subsequent MmeI digestion and self‐ligation results in the production of covalently‐linked paired‐end ditags (PETs) that can be extracted and then concatenated for efficient sequencing. By mapping the PET sequences to assembled genomes, the original DNA fragments from which the PETs were derived can be precisely localized. This unit details two applications of PET technology. In GIS‐PET, ditagging of mRNA converted to full‐length cDNA enables whole‐transcriptome analysis, including novel gene identification, gene prediction validation, and gene expression studies. In ChIP‐PET, ditagging of chromatin immunoprecipitation–enriched genomic DNA fragments enables the global mapping of transcription factor binding sites. A recent innovation (Multiplex Sequencing of Paired‐End ditags; MS‐PET) enables PETs to be sequenced using high‐throughput 454 sequencing, greatly increasing the amount of data that can be collected in each run. Curr. Protoc. Mol. Biol. 79:21.12.1‐21.12.42. © 2007 by John Wiley & Sons, Inc.

Keywords: PET; ditag; paired‐end; mate‐pair; SAGE; DNA sequencing; GIS‐PET; ChIP‐PET; MS‐PET; 454 sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of a GIS Full‐Length cDNA Library
  • Basic Protocol 2: Construction of a ChIP‐DNA Plasmid Library
  • Basic Protocol 3: Construction of a GIS‐PET or ChIP‐PET Library (Ditagging)
  • Alternate Protocol 1: MS‐PET Procedure for High‐Throughput Sequencing
  • Support Protocol 1: Preparation and Validation of pGIS4 and pGIS3h Cloning Vectors
  • Support Protocol 2: Preparation of Adapters
  • Support Protocol 3: PicoGreen Assay for Quantitation of DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction of a GIS Full‐Length cDNA Library

  Materials
  • 2 µg/µl GsuI‐oligo‐dT primer
  • 40 U/µl RNasin‐PLUS RNase inhibitor (Promega)
  • PolyA RNA sample of interest (10 to 20 µg)
  • Nuclease‐free water (Ambion)
  • 2× GC‐I buffer (Takara; available as part of the LA Taq kit)
  • Modified 10 mM dNTP (with 5‐Me‐dCTP instead of dCTP; see recipe)
  • 4.9 M D‐sorbitol (Sigma)
  • 200 U/µl Superscript II reverse transcriptase (RNase H; Invitrogen)
  • Saturated trehalose (see recipe)
  • 20 mg/ml proteinase K (Ambion)
  • 25:24:1 (v/v/v) phenol/chloroform/IAA, pH 6.6 and 7.9 (Ambion)
  • Sodium acetate solutions: 3 M at pH 5.2 ( appendix 22), 1.1 M at pH 4.5, and 1 M at pH 6.1
  • Absolute and 70% ethanol
  • Biotin hydrazide (long arm; Vector Laboratories)
  • Sodium periodate (NaIO 4; Sigma)
  • 10% SDS ( appendix 22),
  • 5 M NaCl
  • Isopropanol
  • 10 U/µl RNaseONE ribonuclease and 10× RNaseONE buffer (Promega)
  • 10 mg/ml yeast tRNA (Ambion)
  • M‐280 streptavidin Dynabeads (Dynal)
  • 1× binding buffer (see recipe)
  • 1× blocking buffer (see recipe)
  • 1× wash buffer (see recipe)
  • Alkaline hydrolysis buffer (see recipe), prepare fresh
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 22)
  • 15 mg/ml GlycoBlue (Ambion)
  • LoTE buffer (optional; see recipe)
  • EB buffer (10 mM Tris⋅Cl, pH 8.5; Qiagen)
  • 0.4 µg/µl GIS‐(N)5 and 0.4 µg/µl GIS‐(N)6 adapters (see Fig. ; validated as in protocol 6)
  • Solutions I (enzyme solution) and II (concatenation buffer) from DNA ligation kit (Takara version 2.1)
  • 5 U/µl ExTaq polymerase and 10× ExTaq buffer with Mg2+ (Takara)
  • 2.5 mM dNTP mix (Takara)
  • 10× Tango buffer (Fermentas)
  • 32 mM S‐adenosyl methionine (SAM; NEB; supplied with MmeI enzyme)
  • 10 mg/ml BSA
  • 5 U/µl GsuI (Fermentas)
  • 10 U/µl NotI (NEB)
  • 1× TEN buffer (see recipe)
  • SYBR Green I (Molecular Probes) or GelStar nucleic acid gel stain (Cambrex BioWhittaker), optional
  • pGIS4 cloning vector cut with NotI/BseRI (obtain from authors; see protocol 5)
  • 5 U/µl T4 DNA ligase and 5× T4 DNA ligase buffer with PEG (Invitrogen)
  • Electrocompetent cells (e.g., OneShot electrocompetent TOP10 cells, Invitrogen)
  • SOC medium (see recipe)
  • LB‐amp agar plates (unit 1.1; use Lennox formulation with 5 g/liter NaCl)
  • 20 µM PMR011 and 20 µM PMR012 PCR primers (Fig. )
  • 0.2‐ and 1.7‐ml thin‐walled PCR tubes
  • Heat/chill block or thermal cycler
  • 1.7‐ml microcentrifuge tubes
  • 1.5‐ml siliconized microcentrifuge tubes
  • 1.5‐ml DNA LoBind tubes, PCR clean, safe lock (Eppendorf or Sigma), optional
  • Magnetic rack for washing Dynabeads (Dynal)
  • Benchtop hot/cold shaking incubator (e.g., Eppendorf)
  • Benchtop rotator (Grant‐bio)
  • Phase Lock Gel Light tubes (Eppendorf), optional
  • cDNA size fractionation columns (Invitrogen)
  • Spectrophotometer with 7‐µl submicro‐volume cell (Amersham) or Nanodrop instrument
  • Electroporator and cuvettes (e.g., BioRad)
  • 15‐ml conical tubes (e.g., Falcon)
  • 96‐well microtiter plates
  • Additional reagents and equipment for phenol extraction and ethanol precipitation (unit 2.1), ligation (units 3.14& 3.16), agarose gel electrophoresis (unit 2.5), restriction enzyme digestion (unit 3.1), DNA quantitation ( 3.NaN), electroporation (unit 9.3), PCR (unit 15.1), and plasmid purification (unit 1.6)

Basic Protocol 2: Construction of a ChIP‐DNA Plasmid Library

  Materials
  • End‐It DNA end‐repair kit (Epicentre) containing:
    • End‐repair enzyme mix: T4 DNA polymerase and T4 polynucleotide kinase
    • End‐repair 10× buffer
    • 2.5 mM dNTP solution
    • 10 mM ATP
  • Sample of interest: ChIP‐enriched genomic DNA fragments (at least 100 ng)
  • 25:24:1 (v/v/v) phenol/chloroform/IAA, pH 7.9 (Ambion)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 70% and absolute ethanol
  • GlycoBlue (Ambion)
  • 1× TEN buffer (see recipe)
  • pGIS3h cloning vector (obtain from authors; prepare as in protocol 5)
  • 5 U/µl T4 DNA ligase and 5× T4 DNA ligase buffer with PEG (Invitrogen)
  • EB buffer (10 mM Tris⋅Cl, pH 8.5; Qiagen)
  • 20 µM PMR011 and 20 µM PMR012 PCR primers (Fig. )
  • Phase Lock Gel Light tubes (Eppendorf), optional
  • Heat/chill block or thermal cycler
  • Additional reagents and equipment for size‐fractionation, transformation, and PCR screening (see protocol 1)

Basic Protocol 3: Construction of a GIS‐PET or ChIP‐PET Library (Ditagging)

  Materials
  • Purified ligation mix (see protocol 1)
  • LB medium (unit 1.1; use Lennox formulation containing 5 g/liter NaCl)
  • HiSpeed Plasmid Maxi kit (Qiagen) or equivalent
  • 2 U/µl MmeI (NEB) and 32 mM S‐adenosyl methionine (SAM; supplied with MmeI)
  • 10× NEBuffer 4 (NEB)
  • pGIS4 cloning vector (obtain from authors; prepare as in protocol 5)
  • Qiaquick gel extraction kit (Qiagen)
  • 10× Tango buffer (Fermentas)
  • 0.1 M DTT
  • 5 to 10 U/µl T4 DNA polymerase (Promega)
  • 10 mM dNTP mix (Invitrogen)
  • 25:24:1 (v/v/v) phenol/chloroform/IAA, pH 7.9 (Ambion)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 15 mg/ml GlycoBlue (Ambion)
  • Absolute and 70% ethanol
  • Solution I (enzyme solution) from DNA ligation kit (Takara version 2.1)
  • EB buffer (10 mM Tris⋅Cl, pH 8.5; Qiagen)
  • Electrocompetent cells (e.g., OneShot electrocompetent TOP10 cells)
  • SOC medium (see recipe)
  • LB‐amp agar plates (unit 1.1; use Lennox formultion with 5 g/liter NaCl)
  • 20 U/µl BamHI and 10× unique BamHI buffer (NEB)
  • 10 mg/ml BSA
  • 1 M MgCl 2
  • LoTE buffer (see recipe)
  • Wide‐range DNA ladder (50 to 10,000 bp; Takara)
  • 4% to 20% PAGE minigel (TBE; 1.0‐mm, 10 wells; Invitrogen)
  • Low DNA mass ladder (Invitrogen)
  • 25‐ and 100‐bp DNA ladders (Invitrogen)
  • 29:1 (v/v) acryamide/bis solution (BioRad)
  • 5× TBE buffer ( appendix 22)
  • TEMED
  • 10% ammonium persulfate (APS)
  • SYBR Green I (Molecular Probes)
  • 7.5 M ammonium acetate stock solution
  • 5 U/µl T4 DNA ligase and 10× ligase buffer with spermidine (see recipe for ligase buffer)
  • Qiaquick PCR purification kit (Qiagen)
  • 6× loading dye (Fermentas)
  • pZErO‐1 cloning vector (Invitrogen)
  • 5× ligase buffer with PEG (Invitrogen)
  • LB agar plates (unit 1.1; use Lennox formulation with 5 g/liter NaCl) with 25 µg/ml Zeocin (Invitrogen)
  • 20 µM PMR011 and PMR012 PCR primers (Fig. )
  • 22 × 22−cm Q‐trays (Genetix) or other large agar plates for bacterial plating
  • Disposable plastic spreaders (e.g., Lazy L‐spreaders, Sigma), optional
  • 500‐ml plastic centrifuge bottles
  • Floor‐standing ultracentrifuge (e.g., Sorvall with an SLA3000 rotor)
  • Phase Lock Gel Light tubes (Eppendorf), optional
  • 0.6‐ and 1.7‐ml microcentrifuge tubes
  • Electroporation cuvettes (0.1‐cm gap; BioRad), prechilled
  • 15‐ml conical tubes (e.g., Falcon)
  • Hot/cold shaking incubator (Eppendorf)
  • Disposable ElutaTubes (Fermentas) or equivalent
  • Dark Reader blue‐light transilluminator (Clare Chemical)
  • 21‐G needles
  • Microspin plastic centrifuge tube filter units (0.45‐µm; Costar Spin‐X or BioRad Mermaid), sterile
  • Nanodrop spectrometer (optional)
  • 96‐well microtiter plates
  • Additional reagents and equipment for plasmid preparation (units 1.6& 1.7), restriction enzyme digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), DNA quantitation (see 21.12 and 3.NaN), phenol extraction and ethanol precipitation (unit 2.1), ligation (units 3.14& 3.16), electroporation (unit 9.3), electroelution (unit 2.6), PAGE (unit 2.7), validation of cloning vector (see 21.12), PCR (unit 15.1)

Alternate Protocol 1: MS‐PET Procedure for High‐Throughput Sequencing

  Materials
  • Single‐PET plasmid DNA from pGIS4‐ or pGIS3h‐derived libraries (see protocol 3, step 18)
  • 10× NEBuffer 2 (NEB)
  • 10 mg/ml BSA
  • 4 U/µl BseRI (NEB)
  • 25:24:1 (v/v/v) phenol/chloroform/IAA, pH 7.9 (Ambion)
  • 3 M sodium acetate, pH 5.2 (Amresco)
  • 15 mg/ml GlycoBlue (Ambion)
  • Isopropanol
  • 70% and absolute ethanol
  • EB buffer (Qiagen)
  • 5 U/µl antarctic phosphatase and 10× buffer (NEB)
  • 20 U/µl BamHI and 10× unique BamHI buffer (NEB)
  • LoTE buffer (see recipe)
  • 7.5 M ammonium acetate
  • 1 M MgCl 2
  • 5 U/µl T4 DNA ligase
  • 10× ligase buffer with spermidine (see recipe)
  • 21‐G needles
  • Phase Lock Gel Light tubes (Eppendorf), optional
  • Disposable ElutaTubes (or equivalent)
  • 0.6‐ and 1.7‐ml microcentrifuge tubes
  • Microspin plastic centrifuge tube filter units (e.g., SpinX or Mermaid; Costar or BIO101)
  • Additional reagents and equipment for restriction enzyme digestion (unit 3.1), phenol extraction and ethanol precipitation (unit 2.5), agarose gel electrophoresis (unit 2.5), ligation (units 3.14& 3.16), and PAGE (unit 2.7)

Support Protocol 1: Preparation and Validation of pGIS4 and pGIS3h Cloning Vectors

  Materials
  • pGIS4 and pGIS3h plasmids (obtain from authors; )
  • HiSpeed plasmid maxi kit (Qiagen)
  • Qiaquick gel extraction kit (Qiagen)
  • 10× NEBuffer 2 and NEBuffer 3 (NEB)
  • 10 U/µl NotI restriction enzyme (NEB)
  • 4 U/µl BseRI restriction enzyme (NEB)
  • 20 U/µl PstI restriction enzyme (NEB)
  • 10 mg/ml BSA (supplied with restriction enzymes)
  • 5‐10 U/µl T4 DNA polymerase (Promega)
  • 10 mM dNTP mix
  • 10× Tango buffer (Fermentas)
  • EB buffer (Qiagen)
  • Compatible DNA stuffers for validation
  • 5 U/µl T4 DNA ligase and 5× T4 DNA ligase buffer with PEG (Invitrogen)
  • Qiaquick PCR purification kit (Qiagen)
  • 5 U/µl Antarctic phosphatase and 10× Antarctic phosphatase buffer (NEB)
  • TE buffer ( appendix 22; optional)
  • Nanodrop spectrometer (optional)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5) and transformation (see protocol 1)

Support Protocol 2: Preparation of Adapters

  Materials
  • Oligonucleotides A and B to be tested
  • 2× TNE buffer (see recipe)
  • PAGE minigel (either 20% or 4% to 20% gradient)
  • NotI enzyme (NEB)
  • 0.2‐ml PCR tubes
  • Thermal cycler

Support Protocol 3: PicoGreen Assay for Quantitation of DNA

  Materials
  • PicoGreen reagent (Invitrogen)
  • TE buffer ( appendix 22)
  • Calf thymus DNA (Sigma‐Aldrich)
  • DNA samples
  • 96‐well, clear flat‐bottom plate (Greiner)
  • Genios (Tecan) or other suitable fluorometer
  • Fluorometer software (e.g., Magellan)
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Figures

Videos

Literature Cited

Literature Cited
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