2019/03/19 by Derek H. Janssens, Steven Henikoff · 1 citation
Biochemistry, Genetics and Molecular Biology · #Advanced biosensing and bioanalysis techniques #Bacterial Genetics and Biotechnology #RNA and protein synthesis mechanisms
paper · pdf · doi:10.17504/protocols.io.zcpf2vn
openalex publication_date 2019/03/19 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/14
We previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein. Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. In the manuscript attached to this version of the protocol, Meers et al. introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility; 2) a modified digestion protocol that prevents premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. Here we provide an updated CUT&RUN protocol that incorporates these enhancements, and provides three different options for the CUT&RUN MNase digestion reaction that are helpful to improve data quality or to increase throughput. 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.