title: Purification of specific chromatin regions using oligonucleotides: capture hybridization analysis of RNA targets (CHART).
abstract: Identification of genomic binding sites and proteins associated with noncoding RNAs will lead to more complete mechanistic characterization of the regulatory activities of noncoding RNAs. Capture hybridization analysis of RNA targets (CHART) is a powerful technique wherein specific RNA molecules are isolated from cross-linked nuclear extracts using complementary, biotinylated capture oligonucleotides, allowing subsequent identification of genomic DNA and proteins cross-linked to the RNA of interest. Here, we describe the procedure for CHART and list strategies to optimize nuclear extract preparation, capture oligonucleotide design, and isolation of nucleic acids and proteins enriched through CHART.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge St., Boston, MA, 02114, USA, cdavis01@fas.harvard.edu.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Davis
authors.forename: Jason A
authors.initials: JA
authors.lastname: West
mesh_terms: D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics / Q000302:isolation & purification; D062085:RNA, Long Noncoding / Q000235:genetics / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D009841:Oligonucleotides; C092595:Pabp2 protein, Drosophila; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; C077885:XIST non-coding RNA