Abstract
We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.
Highlights
The action of transcription factors (TFs) at their binding sites on DNA drives gene expression patterns, and so genome-wide TF mapping has become a central goal both for individual researchers and large-scale infrastructural projects
Chromatin Immuno-Cleavage (ChIC) has the advantage of using TF-specific antibodies to tether Micrococcal Nuclease (MNase) and cleave only at binding sites
The Cleavage Under Targets and Release Using Nuclease (CUT&RUN) modifications of chromatin immunocleavage (ChIC) were motivated by the observation that light MNase treatment of nuclei liberates mononucleosomes and TF-DNA complexes, leaving behind oligonucleosomes (Sanders, 1978; Teves and Henikoff, 2012)
Summary
The action of transcription factors (TFs) at their binding sites on DNA drives gene expression patterns, and so genome-wide TF mapping has become a central goal both for individual researchers and large-scale infrastructural projects. Cells are crosslinked with formaldehyde, chromatin is fragmented and solubilized, an antibody is added, and the antibody-bound chromatin is recovered for DNA extraction. Successive advances in DNA mapping technologies have revolutionized the use of X-ChIP (formaldehyde crosslinking ChIP), and with ChIP-seq, base-pair resolution mapping of TFs became feasible (Rhee and Pugh, 2011; Skene and Henikoff, 2015; He et al, 2015). Improvements to ChIP-seq retain the crosslinking step to preserve the in vivo pattern while the entire genome is fragmented to create a soluble extract for immunoprecipitation. Crosslinking can promote epitope masking and can generate false positive binding sites (Teytelman et al, 2013; Park et al, 2013; Jain et al, 2015; Baranello et al, 2016; Meyer and Liu, 2014)
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