User:Cseibert832/Single cell epigenomics

Single-cell chromatin conformation capture[edit]
Chromosome conformation capture techniques (often abbreviated to 3C technologies or 3C-based methods) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in three dimensional space, but may be separated by many nucleotides in the linear genome.

All 3C methods start with a similar set of steps, performed on a sample of cells. First, the cell genomes are cross-linked, which introduces bonds that "freeze" interactions between genomic loci. The genome is then cut into fragments. Next, random ligation is performed. Lastly, the fragments are sequenced to determine their proximity to each other (fragments are more likely to be ligated to nearby fragments.)

In single-cell 3C, this last step has typically been done using high-throughput sequencing (Hi-C). Although the recovery rate is as low as 2.5% of potential interactions, it has been possible to generate three dimensional maps of entire genomes using this method.

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Single-cell Hi-C[edit]

Chromosome conformation capture techniques (link to Wikipage) (often abbreviated to 3C technologies or 3C-based methods) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in three dimensional space, even if the loci are be separated by kilobases in the linear genome.

Currently, 3C methods start with a similar set of steps, performed on a sample of cells. First, the cells are cross-linked, which introduces bonds between proteins, and between proteins and nucleic acids, that effectively "freeze" interactions between genomic loci. The genome is then digested into fragments through the use of restriction enzymes. Next, proximity based ligation is performed, creating long regions of hybrid DNA[CS2] ,[CS3]  as fragments are more likely to be ligated to nearby fragments. Lastly, the hybrid DNA is sequenced to determine genomic loci that are in close proximity to each other.

Single-cell Hi-C is a modification of the original Hi-C protocol (link to Hi-C in 3C page), which is an adaptation of the 3C method, that allows you to determine proximity of different regions of the genome in a single cell. This method was made possible by performing the digestion and ligation steps in individual nuclei[CS4], as opposed to the original Hi-C protocol, where ligation was performed after cell lysis in a pool containing crosslinked chromatin complexes[CS7]. In single cell Hi-C, after ligation, single cells are isolated and the remaining steps are performed in separate compartments[CS8][CS9], and the hybrid DNA is tagged with a cell-specific barcode. High-throughput sequencing is then performed on the pool of the hybrid DNA from the single cells. Although the recovery rate can be as low as 2.5% of potential interactions, it is possible to generate three dimensional maps of entire genomes using this method [2016 bioarchive paper Cellcycycle]. Additionally, advances have been made in the analysis of Hi-C data,  allowing for the enhancement of HiC datasets to generate even more accurate and detailed contact maps and 3D models [CS10].

[CS3] https://en.wikipedia.org/wiki/Single_cell_epigenomics

[CS4]Nagano, T., Lubling, Y., Stevens, T. J., Schoenfelder, S., Yaffe, E., Dean, W., … Fraser, P. (2013). Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature, 502(7469), 59–64. doi:10.1038/nature12593

[CS7]﻿Lieberman-Aiden E, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009; 326:289–93. [PubMed: 19815776]

[CS8] multiplexed multiwell: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330809/

[CS9]Wells: reuse Nagano paper

[CS10] https://www.nature.com/articles/s41467-018-03113-2

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330809/