ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Autor:	Gera T; Department of Molecular Genetics, Weizmann Institute, Rehovot, Israel., Kumar DK; Department of Molecular Genetics, Weizmann Institute, Rehovot, Israel., Yaakov G; Department of Molecular Genetics, Weizmann Institute, Rehovot, Israel., Barkai N; Department of Molecular Genetics, Weizmann Institute, Rehovot, Israel., Jonas F; School of Science, Constructor University, Bremen, Germany. fjonas@constructor.university.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2846, pp. 263-283.
DOI:	10.1007/978-1-0716-4071-5_16
Abstrakt:	Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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