Open AccessMapping Regulatory Factors by Immunoprecipitation from Native Chromatin
Author(s) -
Orsi Guillermo A.,
Kasinathan Sivakanthan,
Zentner Gabriel E.,
Henikoff Steven,
Ahmad Kami
Publication year - 2015
Publication title -
current protocols in molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.533
H-Index - 42
eISSN - 1934-3647
pISSN - 1934-3639
DOI - 10.1002/0471142727.mb2131s110
Subject(s) - micrococcal nuclease , chromatin immunoprecipitation , immunoprecipitation , chromatin , nuclease , dna , computational biology , genome , chemistry , chip sequencing , dna sequencing , microbiology and biotechnology , biology , genetics , gene , chromatin remodeling , gene expression , nucleosome , promoter
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. © 2015 by John Wiley & Sons, Inc.