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Then the cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000 base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin. [5] Chromatin fragments of 400 - 500bp have proven to be suitable for ChIP assays as they cover two to three nucleosomes.
ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest (transcription factor) binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair.
The chromosome conformation capture (3C) experiment quantifies interactions between a single pair of genomic loci. For example, 3C can be used to test a candidate promoter-enhancer interaction. Ligated fragments are detected using PCR with known primers. [2] [17] That is why this technique requires the prior knowledge of the interacting regions.
Fang et al. have also shown how there are T-ALL specific gain or loss of chromatin insulation, which alters the strength of TAD architecture of the genome, using in situ Hi-C. [81] Low-C has been used to map the chromatin structure of primary B cells of a diffuse large B-cell lymphoma patient and was used to find high chromosome structural ...
ChIP-on-chip requires highly specific antibodies that must recognize its epitope in free solution and also under fixed conditions. If it is demonstrated to successfully immunoprecipitate cross-linked chromatin, it is termed "ChIP-grade". Companies that provide ChIP-grade antibodies include Abcam, Cell Signaling Technology, Santa Cruz, and Upstate.
ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest.
A single sequencing run can scan for genome-wide associations with high resolution, due to the low background achieved by performing the reaction in situ with the CUT&RUN sequencing methodology. ChIP-Seq, by contrast, requires ten times the sequencing depth because of the intrinsically high background associated with the method. [ 6 ]
Also, as was mentioned above, short reads (e.g. 36-50bp from an Illumina Genome Analyzer) represent a part of a sheared fragment when aligned to the genome; therefore, the exact methylation site can fall anywhere within a window that is a function of the fragment size. [19]