While formaldehyde captures the amino and imino groups of both proteins and DNA, the NHS-esters in DSG react with primary amines on proteins and can capture amine-amine interactions. In 2021, Hi-C 3.0 was described by Lafontaine et al., with higher resolution achieved by enhancing crosslinking with formaldehyde followed by disuccinimidyl glutarate (DSG). The key adaptation to the base protocol was the removal of the SDS solubilization step after digestion to preserve nuclear structure and prevent random ligation between fragmented chromatin by ligation within the intact nuclei, which formed the basis of in situ Hi-C. described a Hi-C 2.0 protocol that was able to achieve kilobase (kb) resolution. The use of restriction endonucleases that cut more frequently, or DNaseI and Micrococcal nucleases also significantly increased the resolution of the method. Nevertheless, Hi-C data offered new insights for chromatin conformation as well as nuclear and genomic architectures, and these prospects motivated scientists to put efforts to modify the technique over the past decade.īetween 20, several modifications to the Hi-C protocol have taken place, with 4-cutter digestion or adapted deeper sequencing depth to obtain higher resolution. The Hi-C library also required several days to construct, and the datasets themselves were low in both output and reproducibility. History Īt its inception, Hi-C was a low-resolution, high-noise technology that was only capable of describing chromatin interaction regions within a bin size of 1 million base pairs (Mb). By combining Hi-C data with other datasets such as genome-wide maps of chromatin modifications and gene expression profiles, the functional roles of chromatin conformation in genome regulation and stability can also be delineated. In recent years, Hi-C has found its application in a wide variety of biological fields, including cell growth and division, transcription regulation, fate determination, development, disease, and genome evolution. Īnalyses of Hi-C data not only reveal the overall genomic structure of mammalian chromosomes, but also offer insights into the biophysical properties of chromatin as well as more specific, long-range contacts between distant genomic elements (e.g. As a result, biotin-marked ligation junctions can be purified more efficiently by streptavidin-coated magnetic beads, and chromatin interaction data can be obtained by direct sequencing of the Hi-C library. While 3C focuses on the analysis of a set of predetermined genomic loci to offer “one-versus-some” investigations of the conformation of the chromosome regions of interest, Hi-C enables “all-versus-all” interaction profiling by labeling all fragmented chromatin with a biotinylated nucleotide before ligation. The relative abundance of these chimeras, or ligation products, is correlated to the probability that the respective chromatin fragments interact in 3D space across the cell population. Then, the chromatin is solubilized and fragmented, and interacting loci are re-ligated together to create a genomic library of chimeric DNA molecules. The general procedure of Hi-C involves first crosslinking chromatin material using formaldehyde. Similar to the classic 3C technique, Hi-C measures the frequency (as an average over a cell population) at which two DNA fragments physically associate in 3D space, linking chromosomal structure directly to the genomic sequence. Hi-C comprehensively detects genome-wide chromatin interactions in the cell nucleus by combining 3C and next-generation sequencing (NGS) approaches and has been considered as a qualitative leap in C-technology (chromosome conformation capture-based technologies) development and the beginning of 3D genomics. In general, Hi-C is considered as a derivative of a series of chromosome conformation capture technologies, including but not limited to 3C (chromosome conformation capture), 4C (chromosome conformation capture-on-chip/circular chromosome conformation capture), and 5C (chromosome conformation capture carbon copy). Hi-C (or standard Hi-C) is a high-throughput genomic and epigenomic technique first described in 2009 by Lieberman-Aiden et al. An overview of the Hi-C workflow and its applications in research.
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