User:Ds17b/sandbox

= Chromosome conformation capture =

Experimental methods
All 3C methods start with a similar set of steps, performed on a sample of cells.

First, the cell genomes are cross-linked with formaldehyde, which introduces bonds that "freeze" interactions between genomic loci. Treatment of cells with 1-3% formaldehyde, for 10-30min at room temperature is most common, however, standardization for preventing high protein-DNA cross linking is necessary, as this may negatively affect the efficiency of restriction digestion in the subsequent step. The genome is then cut into fragments with a restriction endonuclease. The size of restriction fragments determines the resolution of interaction mapping. Restriction enzymes (REs) that make cuts on 6bp recognition sequences, such as EcoR1 or HindIII, are used for this purpose, as they cut the genome once every 4000bp, giving ~ 1 million fragments in the human genome. For more precise interaction mapping, a 4bp recognizing RE may also be used. The next step is, random ligation. This takes place at low DNA concentrations in the presence of T4 DNA ligase, such that ligation between cross-linked interacting fragments is favored over ligation between fragments that are not cross-linked. Subsequently, interacting loci are quantified by amplifying ligated junctions by PCR methods.

= Single cell epigenomics = Single cell epigenomics is the study of epigenomics (the complete set of epigenetic modifications on the genetic material of a cell) in individual cells by single cell sequencing. Until recently, bulk cell population measurements were the source for our understanding of epigenetic modifications and correlations. Now, single-cell methods provide understanding of the relationship between epigenetic modifications and gene expression, comparable between cells in an organism. Since 2013, methods have been created including whole-genome single-cell bisulfite sequencing to measure DNA methylation, whole-genome ChIP-sequencing to measure histone modifications, whole-genome ATAC-seq to measure chromatin accessibility and chromosome conformation capture.

Single-cell DNA methylome sequencing
Treatment with bisulfite has drawbacks as it causes nicks and creates abasic sites in the DNA. Using an alternate method, a study by Lorthongpanich et al. combined methylation sensitive restriction enzymes (MSRE) and qPCR to detect failure in DNA-methylation maintenance in mice pre-implantation embryos.

Single-cell ATAC-seq
Tn5-transposase cuts and tags accessible chromatin with sequencing adaptors, in a process known as transposition. These regions can then be sequenced into libraries. In single cell ATAC-seq, cells are first individually captured and assayed under optimized conditions before transposition and library preparation. With primers specific to single cells, these libraries can be amplified for genome wide mapping by high-throughput sequencing. Chromatin accessibility maps can then be generated for individual cells.

Single-cell chromatin conformation capture
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 with T4 DNA ligase is performed with ligating ends marked with a biotinylated nucleotide, enabling streptavidin mediated enrichment of ligated junctions 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, the ligation step takes place within intact cells, following which, individual cells are isolated under the microscope. With indexed primers, libraries are prepared from single cells, and quantified by qPCR. Lastly, using a high-throughput sequencing (Hi-C) pipeline, involving the processing of paired/single end reads, and assessing reproducibility and quality measures, contact read maps are generated.