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

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. © 2016 by John Wiley & Sons, Inc.