Mapping Regulatory Factors by Immunoprecipitation from Native Chromatin

Current Protocols in Molecular Biology - Tập 110 Số 1 - 2015
Guillermo A. Orsi1,2,3, Sivakanthan Kasinathan4,5,3, Gabriel E. Zentner4, Steven Henikoff4,6, Kami Ahmad2
1CNRS–UMR3664/Institut Curie—Centre de Recherche Paris France
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts
3these authors contributed equally to this work
4Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
5Medical Scientist Training Program, University of Washington School of Medicine, Seattle, Washington
6Howard Hughes Medical Institute, Seattle, Washington.

Tóm tắt

AbstractOccupied 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.

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Current Protocols in Molecular Biology

Tập 110 Số 1

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