ChIP‐chip for Genome‐Wide Analysis of Protein Binding in Mammalian Cells

Tae Hoon Kim1, Leah O. Barrera2, Bing Ren3

1 Yale University School of Medicine, New Haven, 2 University of California, San Diego, La Jolla, 3 Ludwig Institute for Cancer Research, La Jolla
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.13
DOI:  10.1002/0471142727.mb2113s79
Online Posting Date:  July, 2007
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ChIP‐chip combines chromatin immunoprecipitation (ChIP) with microarrays (chip) to determine protein‐DNA interactions occurring in living cells. The high throughput nature of this method makes it an ideal approach for identifying transcription factor targets or chromatin modification sites along the genome. UNIT 21.9 describes a protocol for analysis of protein‐DNA interactions in yeast cells. This unit introduces an alternative protocol developed for mammalian cells. Curr. Protoc. Mol. Biol. 79:21.13.1‐21.13.22. © 2007 by John Wiley & Sons, Inc.

Keywords: ChIP‐chip; transcription factor; chromatin modifications; genome tiling microarrays; promoter arrays

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Table of Contents

  • Introduction
  • Basic Protocol 1: ChIP‐Chip Analysis of Protein‐DNA Binding Sites
  • Basic Protocol 2: Analysis of PCR Array Data
  • Support Protocol 1: Preparation of Annealed Linkers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: ChIP‐Chip Analysis of Protein‐DNA Binding Sites

  • Mammalian cells (ATCC # CCL‐186)
  • MEM with Earle's salts (Invitrogen) supplemented with 10% FCS
  • PBS, pH 7.4 (Invitrogen or see recipe)
  • TE buffer, pH 8.0 ( appendix 22)
  • 5 M sodium chloride (NaCl)
  • 0.5 M EDTA, pH 8.0
  • 0.5 M EGTA, pH 8.0
  • 1 M HEPES, pH 8.0
  • 37% formaldehyde
  • 2.5 M glycine
  • Lysis buffer (see recipe)
  • Protein extraction buffer (see recipe)
  • Chromatin extraction buffer (see recipe)
  • Glycerol
  • Dynabeads coupled to appropriate secondary antibodies (Invitrogen)
  • 5 mg/ml BSA in PBS, prepared immediately before use with BSA powder (Sigma‐Aldrich)
  • Antibody directed against protein of interest (e.g., Upstate Biotechnology, Santa Cruz Biotech, Abcam)
  • Triton X‐100
  • 10% sodium deoxycholate (DOC) in distilled water, prepared immediately before use
  • 50× complete protease inhibitor solution, prepared immediately before use by dissolving one tablet (Roche Applied Science) in 1 ml dH 2O
  • RIPA buffer (see recipe)
  • Elution buffer (see recipe)
  • 20 mg/ml glycogen (Roche Applied Science)
  • 20 mg/ml proteinase K
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • Chloroform
  • 75% and 100% ethanol, cold and room temperature
  • 10 mg/ml RNase A (DNase‐free)
  • Qiagen QIAquick PCR purification kit (Qiagen)
  • 3 U/µl T4 DNA polymerase and 10× buffer (New England Biolabs)
  • 10 mg/ml BSA (acetylated, New England Biolabs)
  • 2.5 and 20 mM dNTP mixes (containing 2.5 or 20 mM each of dATP, dCTP, dGTP, and dTTP)
  • 3 M sodium acetate, pH 5.3
  • 400 U/µl T4 DNA ligase and 5× buffer (New England Biolabs)
  • Annealed linkers (see protocol 3)
  • 10× ThermoPol reaction buffer (NEB)
  • 40 mM oJW102 oligo (5′‐GCGGTGACCCGGGAGATCTGAATTC‐3′, HPLC‐purified)
  • 5 U/µl Taq polymerase (Qiagen)
  • 2.5 U/µl PfuUltra polymerase (Stratagene)
  • BioPrime array CGH genomic labeling system kit (Invitrogen) containing:
    • Exo‐Klenow fragment
    • 2.5× random primers solution
    • 10× dCTP nucleotide mix
    • 10× dUTP nucleotide mix
    • Control DNA (salmon sperm)
    • Stop buffer: 0.5 M EDTA, pH 8.0
  • Cy3‐dCTP and Cy5‐dCTP (GE Healthcare Biosciences)
  • Prehybridization solution (see recipe)
  • Reverse osmosis (RO) water
  • Cot‐1 DNA (Invitrogen)
  • 2.2× SSC ( appendix 22)/0.22% SDS, prepare within 1 hr of use
  • Hybridization buffer (see recipe)
  • 0.1× SSC
  • 37°C, 5% CO 2 humidified incubator
  • 500‐cm2 plates (Corning)
  • Cell scraper
  • 250‐ml conical bottles
  • Platform rocker
  • 15‐ and 50‐ml conical tubes
  • Branson 450 sonifier fitted with a microtip
  • Flourometer
  • Magnetic concentrator (Invitrogen)
  • 65°C incubator
  • 1.5‐ml microcentrifuge tubes
  • 12°, 37°, 42°, and 60°C water baths
  • UV spectrophotometer
  • 200‐µl PCR tubes
  • PCR thermal cycler
  • 100°C heating block
  • Coplin jars
  • Cap‐locks for microcentrifuge tubes
  • Tabletop centrifuge (e.g., Eppendorf 5804) with microplate rotor (A‐2‐DWP)
  • Hybridization chambers (Corning)
  • Slide racks
  • Glass staining dish (Wheaton)
  • Scanner (e.g., Axon 4000B)
  • Scanning software (e.g., GenePix)
NOTE: Use only molecular biology grade (DNase‐free) reagents and chemicals.

Basic Protocol 2: Analysis of PCR Array Data

  • 40 mM oJW102 (5′‐GCGGTGACCCGGGAGATCTGAATTC‐3′, HPLC‐purified)
  • 40 mM oJW103 (5′‐GAATTCAGATC‐3′, HPLC‐purified)
  • 1 M Tris·Cl, pH 7.9 ( appendix 22)
  • 1.5‐ml microcentrifuge tubes
  • 70° and 95°C heating blocks
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