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ChIP‐Seq: A Method for Global Identification of Regulatory Elements in the Genome

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Abstract
Table of Contents
Materials
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Abstract

This unit describes ChIP?Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high?throughput sequencing (Seq), and enables the genome?wide identification of binding sites of transcription factors (TFs) and other DNA?binding proteins. The process is initiated by cross?linking DNA and DNA?bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA?binding protein is then used to immunoprecipitate specific DNA?TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30? to 35?nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites. Curr. Protoc. Mol. Biol. 91:21.19.1?21.19.14. © 2010 by John Wiley & Sons, Inc.

Keywords: mammalian cells; ChIP; ChIP?Seq; binding site; genome?wide

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Introduction
Strategic Planning
Basic Protocol 1: Preparation of ChIP DNA
Basic Protocol 2: Sequencing ChIP DNA Using Illumina Genome Analyzer
Support Protocol 1: Assess Quality of ChIP DNA
Reagents and Solutions
Commentary
Literature Cited
Figures

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Basic Protocol 1: Preparation of ChIP DNA Materials

Cells growing in appropriate growth medium (see appendix 3F )
37% formaldehyde
2 M glycine (see recipe )
1× PBS with protease inhibitors: dissolve one Complete Protease Inhibitor tablet (Roche) in 22.5 ml ice‐cold water, add 2.5 ml 10× PBS ( appendix 22 ) and vortex to dissolve; keep on ice
Hypotonic buffer (see recipe ), ice‐cold
1× RIPA buffer (see recipe )
Antibody for immunoprecipitation
Protein A–Agarose, Fast Flow (Millipore)
Elution Buffer 1 (see recipe )
Elution Buffer 2 (see recipe )
TE buffer ( appendix 22 )
20 mg/ml proteinase K
RNase (Qiagen, cat. no. 19101)
QIAquick PCR purification kit (Qiagen) including QIAquick purification columns and elution buffer (Buffer EB)
Nuclease‐free water (for preparing reagents listed above)

15‐ and 50‐ml conical tubes (Falcon)
Clay Adams Nutator mixer
7‐ml Dounce homogenizer (Kontes Glass)
2‐ml microcentrifuge tubes, prechilled
Probe sonicator: e.g., Branson 250 Sonifier
65° and 45°C water baths

Additional reagents and equipment for basic cell culture techniques (including trypsinization; appendix 3F ) and immunoprecipitation (unit 10.16 )

NOTE: Use nuclease‐free water in all recipes and protocol steps.NOTE: Vary the volume of each reagent depending upon the number of ChIP DNAs in the experiment.NOTE: The tissue culture cells need to be maintained in appropriate medium and in growth conditions optimal for healthy cells. Grow cells in freshly prepared medium, change medium every 2 to 3 days to avoid overgrowth of cells, and plate cells at appropriate density when passaging cells. Also see appendix 3F . Basic Protocol 2: Sequencing ChIP DNA Using Illumina Genome Analyzer Materials

ChIP DNA sample from step 26 of protocol 1
End‐It DNA End Repair Kit (Epicentre) containing:
- 2.5 mM dNTP Mix
- 10 mM ATP
- End‐Repair Enzyme Mix
- End‐Repair 10× Buffer
QIAquick PCR Purification Kit (Qiagen) including QIAquick columns and elution buffer (Buffer EB)
100 mM dATP (New England Biolabs)
Klenow fragment of DNA polymerase I (3′→5′ exo minus; New England Biolabs, cat. no. M0212) and NEBuffer 2 (New England Biolabs)
LigaFast Ligase and 2× DNA ligase buffer (Promega)
Illumina Adapter Oligo Mix
QIAquick MinElute columns (Qiagen)
2% agarose E‐gel (Invitrogen) with SYBR SAFE gel stain (also see unit 2.5 for general agarose gel electrophoresis protocols)
Qiagen Gel Extraction Kit
Illumina PCR Primers 1.1 and 2.1
2× Phusion HF Master Mix (New England Biolabs, cat. no. F531)

Illumina Genome Analyzer with flowcells, reagents, and cluster station
Eland data collection software (Illumina)

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

NOTE: Use nuclease‐free water in all recipes and protocol steps. Support Protocol 1: Assess Quality of ChIP DNA Materials

Input DNA from step 26 of protocol 1
ChIP DNA prepared using TF‐specific antibody ( protocol 1 )
ChIP DNA prepared using normal IgG ( protocol 1 )
Taq Master Mix (Qiagen)
SYBR Mix for qPCR (Roche)
Control Pol II primers:
- 1. (FWD) 5′GGAAAGAGTGGTGCGGTAGC‐3′
- (RVS) 5′‐GGGATGTTCACCACCTGCTT‐3′
- 2. (FWD) 5′‐CTGAGGGCCACGGAAGAAA‐3′
- (RVS) 5′‐TGCGTGGAACAACGGTAGC‐3′

Additional reagents and equipment for agarose gel electrophoresis (unit 2.5 ) or qPCR (unit 15.8 )

NOTE: Use nuclease‐free water in all recipes and protocol steps.

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Figure 21.19.1 Schematic diagram of the ChIP‐Seq process.

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Figure 21.19.2 Evaluation of ChIP DNA quality by PCR. 1/50th of ChIP DNA prepared from 5 × 107 cells was assayed for enrichment of Pol II ChIP DNA at the promoter site of two target genes.

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Figure 21.19.3 Signal map of Pol II ChIP DNA. (A ) Signal map of two biological replicates of Pol II ChIP DNA and control IgG ChIP DNA. Enrichment of Pol II ChIP DNA, but not IgG ChIP DNA, is shown over an entire chromosome. (B ) Illustrates a close‐up view of Pol II binding near the transcription start site (TSS) of a RefSeq gene.

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Literature Cited

Literature Cited
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	Gilmour, D.S. and Lis, J.T. 1984. Detecting protein‐DNA interactions in vivo: Distribution of RNA polymerase on specific bacterial genes. Proc. Natl. Acad. Sci. U.S.A. 81:4275‐4279.
	Iyer, V.R., Horak, C.E., Scafe, C.S., Botstein, D., Snyder, M., and Brown, P.O. 2001. Genomic binding sites of the yeast cell‐cycle transcription factors SBF and MBF. Nature 409:533‐538.
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	Rozowsk, J., Euskirchen, G., Auerbach, R.K., Zhang, Z.D., Gibson, T., Bjornson, R., Carriero, N., Snyder, M., and Gerstein, M.B. 2009. PeakSeq enables systematic scoring of ChIP‐seq experiments relative to controls. Nat. Biotechnol. 27:66‐75.
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	Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nussbaum, C., Myers, R.M., Brown, M., Li, W., and Liu, S. 2008. Model‐based analysis of ChIP‐Seq (MACS). Genome Biol. 9:R137.

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