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.
The plasmid pAG-ERH-MNase-6xHIS-HA is available from Addgene. Sequencing datasets are available from GEO (GSE126612).
A streamlined protocol and analysis pipeline for CUT&RUN chromatin profilingNCBI Gene Expression Omnibus, GSE126612.
- Michael P Meers
- Terri D Bryson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Stephen Parker, University of Michigan, United States
© 2019, Meers et al.
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