ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy

Abstract

This unit describes the ChIP-exo methodology, which combines chromatin immunoprecipitation (ChIP) with lambda exonuclease digestion followed by high-throughput sequencing. ChIP-exo allows identification of a nearly complete set of the binding locations of DNA-binding proteins at near-single-nucleotide resolution with almost no background. The process is initiated by cross-linking DNA and associated proteins. Chromatin is then isolated from nuclei and subjected to sonication. Subsequently, an antibody against the desired protein is used to immunoprecipitate specific DNA-protein complexes. ChIP DNA is purified, sequencing adaptors are ligated, and the adaptor-ligated DNA is then digested by lambda exonuclease, generating 25- to 50-nucleotide fragments for high-throughput sequencing. The sequences of the fragments are mapped back to the reference genome to determine the binding locations. The 5' ends of DNA fragments on the forward and reverse strands indicate the left and right boundaries of the DNA-protein binding regions, respectively.

Figure 1

Scheme for ChIP-exo. After ChIP, 3′ sonicated ends are demarcated from the eventual exonuclease-treated 5′ end, by ligating P2-T Adaptor to the ChIP DNA on the resin prior to exonuclease digestion. 5′ to 3′ exonuclease trimming up to the site of cross-linking selectively eliminates the P2-T adaptor sequence attached at the 5′ end of each strand. After crosslink reversal, eluted single-stranded DNA is made double-stranded by P2 PCR Primer extension. Then, a P1-T Adaptor was ligated to the exonuclease-treated end. The resulting library is subjected to high-throughput sequencing. Mapping the 5′ ends of the resulting sequencing tags to the reference genome demarcates the exonuclease barrier and thus the precise site of protein-DNA cross-linking (Rhee and Pugh, 2011).