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Nat Protoc. 2013;8(7):1337-53. doi: 10.1038/nprot.2013.077. Epub 2013 Jun 13.

Genome-wide measurement of protein-DNA binding dynamics using competition ChIP.

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  • 1Department of Biology, Carolina Center for the Genome Sciences, Curriculum in Genetics and Molecular Biology and Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, USA.

Abstract

Competition chromatin immunoprecipitation (competition ChIP) enables experimenters to measure protein-DNA dynamics at a single locus or across the entire genome, depending on the detection method. Competition ChIP relies on a cell containing two copies of a single DNA-associated factor, with each copy of the factor differentially epitope tagged. One of the copies is expressed constitutively and the second is induced as a competitor. The ratio of isoforms associated with discrete genomic locations is detected by ChIP-on-chip (ChIP-chip) or ChIP-sequencing (ChIP-seq). The rate at which the resident isoform of the protein is replaced by the competitor at each binding location enables the calculation of residence time for that factor at each site of interaction genome wide. Here we provide a detailed protocol for designing and performing competition ChIP experiments in Saccharomyces cerevisiae, which takes ∼5 d to complete (not including strain production and characterizations, which may take as long as 6 months). Included in this protocol are guidelines for downstream bioinformatic analysis to extract residence times throughout the genome.

PMID:
23764940
[PubMed - indexed for MEDLINE]
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