Chromatin Interaction Analysis Using Paired‐End Tag Sequencing

Melissa J. Fullwood1, Yuyuan Han1, Chia‐Lin Wei1, Xiaoan Ruan1, Yijun Ruan1

1 Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.15
DOI:  10.1002/0471142727.mb2115s89
Online Posting Date:  January, 2010
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Abstract

Chromatin Interaction Analysis using Paired‐End Tag sequencing (ChIA‐PET) is a technique developed for large‐scale, de novo analysis of higher‐order chromatin structures. Cells are treated with formaldehyde to cross‐link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation. The Paired‐End Tag (PET) strategy is applied to the construction of ChIA‐PET libraries, which are sequenced by high‐throughput next‐generation sequencing technologies. Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome‐wide map of binding sites and chromatin interactions mediated by the protein factor under study. This unit describes ChIA‐PET for genome‐wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies. Curr. Protoc. Mol. Biol. 89:21.15.1‐21.15.25. © 2010 by John Wiley & Sons, Inc.

Keywords: PET; paired‐end; mate‐pair; SAGE; DNA sequencing; ChIA‐PET; 454 sequencing; Illumina sequencing; transcription factor binding sites; chromatin interactions; chromosome conformation capture; chromatin immunoprecipitation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of a ChIA‐PET Library
  • Support Protocol 1: Preparation of Linkers and Adapters for ChIA‐PET
  • Support Protocol 2: Validation of Linkers for ChIA‐PET
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of a ChIA‐PET Library

  Materials
  • ChIP DNA bound to Sepharose beads (unit 21.3)
  • TE buffer, pH 8.0 ( appendix 22)
  • Elution buffer (see recipe)
  • 20 mg/ml proteinase K (Invitrogen)
  • Buffer EB (Qiagen)
  • 10× T4 polymerase buffer (Promega)
  • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
  • Nuclease‐free H 2O (see unit 4.1)
  • 9.7 U/µl T4 DNA polymerase (Promega)
  • Wash buffer (see recipe)
  • 200 ng/µl biotinylated half‐linkers A and B (Fig. ; see Support Protocols protocol 31 and protocol 42)
  • 5× T4 DNA ligase buffer with PEG (Invitrogen)
  • 30 U/µl T4 DNA ligase (Fermentas)
  • 10× T4 DNA ligase buffer (without PEG; New England Biolabs)
  • 10 U/µl T4 DNA polynucleotide kinase (New England Biolabs)
  • Elution buffer (see recipe)
  • 20% (v/v) Triton X‐100
  • 25:24:1 phenol/chloroform/isoamyl alcohol, pH 7.9 (Ambion)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 15 mg/ml GlycoBlue (Ambion)
  • Isopropanol
  • 75% ethanol
  • 10× NEBuffer 4 (New England Biolabs)
  • 10× (500 µM) S‐adenosylmethionine (SAM; New England Biolabs), freshly prepared, by adding 1 µl 640× SAM from the manufacturer to 63 µl H 2O
  • 200 ng/µl nonbiotinylated linker (Fig. ; Support Protocols protocol 31 and protocol 42)
  • 2 U/µl MmeI restriction endonuclease (New England Biolabs)
  • Dynabeads M‐280 Streptavidin (Invitrogen)
  • 1× and 2× B&W buffer (see recipe)
  • 200 ng/µl 454 GS20 Adapters A and B (Fig. ; see Support Protocols protocol 31 and protocol 42)
  • 10× NEBuffer 2 (New England Biolabs)
  • 10 U/µl E. coli DNA polymerase I (New England Biolabs)
  • 10 µM 454 PCR Primer A and biotinylated Primer B (Fig. )
  • HotStarTaq Master Mix (Qiagen)
  • 6% TBE PAGE gel (5 wells; Invitrogen, by special request only; also see unit 2.7)
  • 10× TBE buffer ( appendix 22)
  • SYBR Green I (Invitrogen Molecular Probes)
  • QIAquick PCR Purification Kit (Qiagen)
  • 25 µM Illumina 1‐454 primer (Fig. )
  • 25µM Illumina 2‐454 primer (Fig. )
  • Phusion High‐Fidelity PCR Master Mix with HF buffer (Finnzymes; http://www.finnzymes.us/)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Intelli‐Mixer RM‐2L (Palico Biotech, http://www.palicobio.com/)
  • 2‐ml Phase‐Lock Gel tubes (Eppendorf)
  • Microspin plastic centrifuge tube filter units (0.45‐µm, e.g., Corning Costar Spin‐X)
  • 50‐ml MaXtract High Density tubes (Qiagen)
  • 50‐ml polypropylene copolymer (PPCO) centrifuge tubes (Nalgene)
  • Magnetic Particle Collector (Invitrogen)
  • 1.5‐ml DNA LoBind Tubes (Eppendorf)
  • Thermal cycler
  • 0.2‐ml PCR tubes
  • Dark Reader Transilluminator (Clare Chemical Research; http://www.clarechemical.com/)
  • 0.6‐ml microcentrifuge tubes pierced at bottom with 21‐G needle
  • Agilent 2100 Bioanalyzer (Agilent Technologies)
  • Agilent DNA 1000 Kit (Agilent Technologies)
  • Additional reagents and equipment for phenol/chloroform extraction and isopropanol precipitation of DNA (unit 2.1), PicoGreen dsDNA quantitation ( appendix 3D and unit 21.12), PCR (unit 15.1), and analysis of PCR products by electrophoresis (unit 2.7)

Support Protocol 1: Preparation of Linkers and Adapters for ChIA‐PET

  Materials
  • Linker and adapter oligonucleotides (Fig. )
  • 1× TNE buffer (see recipe)
  • 4% to 20% TBE PAGE gel (10 wells; Invitrogen)
  • 25‐bp DNA ladder
  • Additional reagents and equipment for annealing of oligonucleotides (unit 21.12, Support Protocol 2) and electrophoresis of DNA (unit 2.7)

Support Protocol 2: Validation of Linkers for ChIA‐PET

  Materials
  • DNA fragment X of known size (blunt‐ended, 5′‐phosphorylated, ∼1 to 3 kb)
  • 200 ng/µl annealed biotinylated half‐linkers A and B (Fig. )
  • 5× T4 DNA ligase buffer with PEG (Invitrogen)
  • 30 U/µl T4 DNA ligase (Fermentas)
  • Nuclease‐free H 2O (see unit 4.1)
  • QIAquick PCR Purification Kit (Qiagen)
  • Buffer EB (Qiagen)
  • 10× T4 DNA ligase buffer (without PEG; New England Biolabs)
  • 10 U/µl T4 DNA polynucleotide kinase (New England Biolabs)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 15 mg/ml GlycoBlue (Ambion)
  • Isopropanol
  • 75% ethanol
  • 10× NEBuffer 4 (New England Biolabs)
  • 10× (500 µM) S‐adenosylmethionine (SAM; New England Biolabs), freshly prepared by adding 1 µl 640× buffer from the manufacturer to 63 µl H 2O
  • 200 ng/µl nonbiotinylated linker (Fig. ; Support Protocols protocol 31 and protocol 42)
  • 2 U/µl MmeI restriction endonuclease (New England Biolabs)
  • Dynabeads M‐280 Streptavidin (Invitrogen)
  • 1× and 2× B&W buffer (see recipe)
  • 200 ng/µl 454 GS20 Adapters A and B (Fig. ; see Support Protocols protocol 31 and protocol 42)
  • 10× NEBuffer 2 (New England Biolabs)
  • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, and dTTP)
  • 10 U/µl E. coli DNA polymerase I (New England Biolabs)
  • 10 µM 454 PCR Primer A and biotinylated Primer B (Fig. )
  • HotStarTaq Master Mix (Qiagen)
  • 4% to 20% TBE PAGE gel (10 wells; Invitrogen)
  • Intelli‐Mixer RM‐2L (Palico Biotech, http://www.palicobio.com/)
  • Magnetic Particle Collector (Invitrogen)
  • 1.5‐ml DNA LoBind tubes (Eppendorf)
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for phenol/chloroform extraction of DNA (unit 2.1), PCR (unit 15.1), and analysis of PCR products by electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

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