Interaction between transcription regulatory regions of prolactin chromatin. The spatial organization of the human genome. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.Ĭremer, T. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions.
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