title: Mapping regulatory factors by immunoprecipitation from native chromatin.
abstract: Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.|CNRS-UMR3664/Institut Curie-Centre de Recherche, Paris, France.|These authors contributed equally to this work.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Orsi
authors.affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.|Medical Scientist Training Program, University of Washington School of Medicine, Seattle, Washington.|These authors contributed equally to this work.
authors.forename: Sivakanthan
authors.initials: S
authors.lastname: Kasinathan
authors.affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.
authors.forename: Gabriel E
authors.initials: GE
authors.lastname: Zentner
authors.affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.|Howard Hughes Medical Institute, Seattle, Washington.
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Kami
authors.initials: K
authors.lastname: Ahmad
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D046911:Macromolecular Substances / Q000302:isolation & purification; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002843:Chromatin; D046911:Macromolecular Substances; D014157:Transcription Factors
keywords: MNase-seq; ORGANIC; chromatin; transcription; transcription factor
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