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URL: https://dx.doi.org/10.17504/protocols.io.mgjc3un

Authors: Peter J. Skene, Steven Henikoff

Group: Human Cell Atlas Method Development Community

Summary: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.In summary, CUT&RUN has several advantages over ChIP-seq: (1) The method is performed in situ in non-crosslinked cells and does not require chromatin fragmentation or solubilization; (2) The intrinsically low background allows low sequence depth and identification of low signal genomic features invisible to ChIP; (3) The simple procedure can be completed within a day and is suitable for robotic automation; (4) The method can be used with low cell numbers compared to existing methodologies; (5) A simple spike-in strategy can be used for accurate quantitation of protein-DNA interactions. As such, CUT&RUN represents an attractive replacement for ChIPseq, which is one of the most popular methods in biological research.

Affiliations: Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington, USA 98109, Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington, USA 98109

External URL: https://www.biorxiv.org/content/early/2017/09/24/193219

Version: 1

Publication Date: 2018

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