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Curr Protoc Plant Biol. 2016;1(2):293-306. doi: 10.1002/cppb.20014. Epub 2016 Jun 10.

Profiling of transcription factor binding events by chromatin immunoprecipitation sequencing (ChIP-seq).

Author information

1
Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
2
Genomic Analysis Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
3
Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Abstract

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.

KEYWORDS:

Arabidopsis; ChIP-seq; transcription factor

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