Program in Systems Biology; Department of Biochemistry and Molecular Pharmacology; Graduate School of Biomedical Sciences
Amino Acids, Peptides, and Proteins | Computational Biology | Enzymes and Coenzymes | Genomics | Molecular Biology | Structural Biology
The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.
Hi-C, chromatin interactions, chromosome conformation capture, cross-linking, restriction enzyme
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Copyright © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
DOI of Published Version
Lafontaine DL, Yang L, Dekker J, Gibcus JH. Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture. Curr Protoc. 2021 Jul;1(7):e198. doi: 10.1002/cpz1.198. PMID: 34286910; PMCID: PMC8362010. Link to article on publisher's site
Lafontaine DL, Yang L, Dekker J, Gibcus JH. (2021). Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture. Open Access Publications by UMass Chan Authors. https://doi.org/10.1002/cpz1.198. Retrieved from https://escholarship.umassmed.edu/oapubs/4833
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