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Molecular mechanism of EPC1-PHF1 fusion protein (CUT and RUN-seq Homo Sapiens)

ABSTRACT: Molecular mechanism of EPC1-PHF1 fusion protein (CUT and RUN-seq Homo Sapiens)

PROVIDER: PRJNA807169 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18034619_1.fastq.gz	Fastqsanger.gz
	SRR18034619_2.fastq.gz	Fastqsanger.gz
	SRR18034620_1.fastq.gz	Fastqsanger.gz
	SRR18034620_2.fastq.gz	Fastqsanger.gz
	SRR18034621_1.fastq.gz	Fastqsanger.gz

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Molecular mechanism of EPC1-PHF1 fusion protein

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Project description:Over the past two decades, the dominant technology for chromatin profiling has been chromatin immunoprecipitation (ChIP), in which cellular components are solubilized to fragment chromatin, and an antibody is added to precipitate a DNA/protein complex of interest. We previously described a novel alternative to ChIP, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which the antibody is added to permeabilized cells followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein to the antibody (PMID 29651053). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four extensions to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that allows simplified purification using a commercial kit; 2) a modified digestion protocol that prevents release of the enzyme and so minimizes artifactual cleavage of accessible DNA; 3) a calibration strategy based on carryover of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy that is customized for the low-background profiles obtained using CUT&RUN. 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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