High‐Resolution Chromatin Profiling Using CUT&RUN. Issue 1 (28th January 2019)

• Record Type: Journal Article
• Authors: Hainer, Sarah J.; Fazzio, Thomas G.
• Editors: Ausubel, Frederick M.; Brent, Roger; Kingston, Robert E.; Moore, David D.; Seidman, J.G.; Smith, John A.; Struhl, Kevin
• Is Part Of: Current protocols in molecular biology. Volume 126:Issue 1(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-01-28
• Subjects: chromatin -- CUT&RUN -- protein A–micrococcal nuclease -- transcription factor; Molecular biology -- Technique -- Periodicals; Molecular biology -- Laboratory manuals; Molecular Biology -- methods; Biologie moléculaire -- Technique; Biologie moléculaire -- Manuels de laboratoire; Molecular biology; Molecular biology -- Technique; Laboratory Manual; Electronic reference sources; Laboratory manuals; 572.8028
• DOI: 10.1002/cpmb.85
• Languages: English
• ISSNs: 1934-3639

Abstract

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. © 2019 by John Wiley & Sons, Inc.