title: Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP.
abstract: The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.
authors: Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University.
authors: Toshitsugu
authors: T
authors: Fujita
authors: Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University; hodaka@biken.osaka-u.ac.jp.
authors: Hodaka
authors: H
authors: Fujii
mesh_terms: D047369:Chromatin Immunoprecipitation / Q000379:methods*; D016678:Genome; D023281:Genomics / Q000379:methods*