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Improved CUT&RUN chromatin profiling tools.

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.

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  • Authors+Show Affiliations

    ,

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States.

    ,

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States. Howard Hughes Medical Institute, United States.

    ,

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States.

    Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States. Howard Hughes Medical Institute, United States.

    Source

    eLife 8: 2019 Jun 24 pg

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    31232687

    Citation

    Meers, Michael P., et al. "Improved CUT&RUN Chromatin Profiling Tools." ELife, vol. 8, 2019.
    Meers MP, Bryson TD, Henikoff JG, et al. Improved CUT&RUN chromatin profiling tools. Elife. 2019;8.
    Meers, M. P., Bryson, T. D., Henikoff, J. G., & Henikoff, S. (2019). Improved CUT&RUN chromatin profiling tools. ELife, 8, doi:10.7554/eLife.46314.
    Meers MP, et al. Improved CUT&RUN Chromatin Profiling Tools. Elife. 2019 Jun 24;8 PubMed PMID: 31232687.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Improved CUT&RUN chromatin profiling tools. AU - Meers,Michael P, AU - Bryson,Terri D, AU - Henikoff,Jorja G, AU - Henikoff,Steven, Y1 - 2019/06/24/ PY - 2019/02/22/received PY - 2019/06/22/accepted PY - 2019/6/25/pubmed PY - 2019/6/25/medline PY - 2019/6/25/entrez KW - chromatin KW - chromosomes KW - epigenomics KW - gene expression KW - genetics KW - genomics KW - human KW - spike-in calibration JF - eLife JO - Elife VL - 8 N2 - 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. SN - 2050-084X UR - https://www.unboundmedicine.com/medline/citation/31232687/Improved_CUTRUN_chromatin_profiling_tools L2 - https://doi.org/10.7554/eLife.46314 DB - PRIME DP - Unbound Medicine ER -