title: 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.
authors.affiliation: Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Ho Sung
authors.initials: HS
authors.lastname: Rhee
authors.forename: B Franklin
authors.initials: BF
authors.lastname: Pugh
mesh_terms: D000818:Animals; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins