Profiling of Transcription Factor Binding Events by Chromatin Immunoprecipitation Sequencing (ChIP‐seq)

Liang Song1, Yusuke Koga1, Joseph R. Ecker2

1 Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, 2 Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20014
Online Posting Date:  June, 2016
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In multi‐cellular organisms, gene expression is orchestrated by thousands of transcription factors (TF). Chromatin immunoprecipitation followed by sequencing (ChIP‐seq) is a robust tool to investigate gene expression because this technique profiles in vivo protein‐DNA interaction at a genome‐wide scale. Eight years after the first ChIP‐seq paper, there are limited reports of ChIP‐seq experiments in plants, especially for sequence‐specific DNA binding TFs. This lag greatly prevents our understanding of transcriptional regulation in an entire kingdom. In order to bridge the technical gap, we describe a ChIP‐seq procedure that we have successfully applied to dozens of sequence‐specific DNA binding TFs. The basic protocol includes procedures to isolate nuclei, sonicate chromatin, immunoprecipitate TF‐DNA complex, and recover ChIP‐enriched DNA fragments. The support protocol also describes practices to optimize library preparation by a gel‐free DNA size selection. Lastly, examples are given to optimize library amplification using real‐time PCR. © 2016 by John Wiley & Sons, Inc.

Keywords: Arabidopsis; ChIP‐seq; transcription factor

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Table of Contents

  • Introduction
  • Basic Protocol 1: Enrichment of Transcription Factor Bound DNA Fragments by Chromatin Immunoprecipitation
  • Support Protocol 1: Optimization of ChIP‐seq Library Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Enrichment of Transcription Factor Bound DNA Fragments by Chromatin Immunoprecipitation

  • Sample material: Arabidopsis tissues
  • Fixation buffer (see recipe)
  • Formaldehyde (37% in H 2O; Sigma Aldrich)
  • 2 M glycine
  • 10 mM HEPES‐NaOH, pH 7.4
  • Liquid nitrogen
  • Dynabeads protein G/Dynabeads protein A (Thermo Fisher Scientific)
  • PBS‐BSA (see recipe)
  • ChIP‐grade antibody (10 μg per ChIP)
  • Extraction buffer I (see recipe)
  • Extraction buffer II (see recipe)
  • Extraction buffer III (see recipe)
  • Nuclei lysis buffer (see recipe)
  • ChIP dilution buffer (see recipe)
  • Low salt wash buffer (see recipe)
  • High salt wash buffer (see recipe)
  • Final wash buffer (see recipe)
  • Elution buffer (see recipe)
  • Proteinase K (20 mg/ml, Thermo Fisher Scientific)
  • 25:24:1 Phenol:chloroform:isoamyl alcohol, pH 8.0
  • 5 M NaCl
  • 100% ethanol
  • Glycogen (5 mg/ml)
  • Vacuum pump (ME 4 NT diaphragm pump, VACUUBRAND; maximum pumping speed 4.0/4.4 m3/hr)
  • DynaMag‐2 Magnet (Thermo Fisher Scientific)
  • Tube rotator
  • Mortar
  • Pestle
  • 50‐ml conical centrifuge tubes
  • Cheesecloth
  • Miracloth (EMD Millipore)
  • Low‐binding DNase‐ and RNase‐free microcentrifuge tubes and pipet tips
  • Refrigerated centrifuge for 50 ml tubes
  • Refrigerated microcentrifuge
  • Bioruptor (Diagenode)
  • Vortex mixer
  • Magnet
  • Thermomixer (Eppendorf)
  • Phase Lock Gel heavy (5 PRIME) or MaXtract High Density (Qiagen)
  • −80°C freezer
  • Qubit fluorometer (Thermo Fisher Scientific)/TapeStation (Agilent Technologies)
  • Desiccator with a stopcock (Bel‐Art)
  • Spatula
NOTE: Use RNase‐ and DNase‐free reagents, deionized distilled water, and low binding plasticware, and barrier pipet tips throughout the experiment.

Support Protocol 1: Optimization of ChIP‐seq Library Preparation

  Additional Materials
  • ChIP‐enriched DNA, a few nanograms or more (see the Basic Protocol)
  • AMPure XP beads (Beckman Coulter)
  • 80% (v/v) ethanol, freshly prepared
  • 1.5‐ml microcentrifuge tube
  • Vortex mixer
  • DynaMag‐2 Magnet (Thermo Fisher Scientific)
  • Thermomixer (Eppendorf)
  • TapeStation
NOTE: Use RNase‐ and DNase‐free reagents, deionized distilled water, and low binding plasticware, and barrier pipette tips throughout the experiment.
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Literature Cited

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