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Curr Protoc Mol Biol. 2019 Apr;126(1):e85. doi: 10.1002/cpmb.85. Epub 2019 Jan 28.

High-Resolution Chromatin Profiling Using CUT&RUN.

Author information

1
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania.
2
Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts.

Abstract

Determining the genomic location of DNA-binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein-DNA interactions at high resolution. In CUT&RUN, a recombinant protein A-microccocal nuclease (pA-MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde-crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA-sequencing libraries that can be sequenced in pools of at least 30. Therefore, CUT&RUN provides an alternative to ChIP-seq approaches for mapping chromatin proteins, which typically have relatively high signal-to-noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA-sequencing libraries, and analyzing the resulting datasets.

KEYWORDS:

CUT&RUN; chromatin; protein A-micrococcal nuclease; transcription factor

PMID:
30688406
PMCID:
PMC6422702
[Available on 2020-04-01]
DOI:
10.1002/cpmb.85
[Indexed for MEDLINE]

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