1. Chromosomes and Gene Expression
  2. Genetics and Genomics
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Improved CUT&RUN chromatin profiling tools

  1. Michael P Meers
  2. Terri D Bryson
  3. Jorja G Henikoff
  4. Steven Henikoff  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
Research Advance
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Cite this article as: eLife 2019;8:e46314 doi: 10.7554/eLife.46314

Abstract

Previously we described a novel alternative to Chromatin Immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

Data availability

The plasmid pAG-ERH-MNase-6xHIS-HA is available from Addgene. Sequencing datasets are available from GEO (GSE126612).

The following data sets were generated

Article and author information

Author details

  1. Michael P Meers

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Terri D Bryson

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jorja G Henikoff

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Steven Henikoff

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States
    For correspondence
    steveh@fhcrc.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7621-8685

Funding

Howard Hughes Medical Institute

  • Michael P Meers

National Institutes of Health (4DN TCPA A093)

  • Terri D Bryson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Stephen Parker, University of Michigan, United States

Publication history

  1. Received: February 22, 2019
  2. Accepted: June 22, 2019
  3. Accepted Manuscript published: June 24, 2019 (version 1)
  4. Version of Record published: June 28, 2019 (version 2)

Copyright

© 2019, Meers et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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