High‐Resolution Chromatin Profiling Using CUT&RUN. Issue 1 (28th January 2019) - Hainer, Sarah J.
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.