EXPERIMENT_TITLE: GSM2795536: Rao-2017-HIC002; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were crosslinked and then lysed with nuclei permeabilized but still intact. DNA was then restricted and the overhangs filled in incorporating a biotinylated base. Free ends were then ligated together in situ. Crosslinks were reversed, the DNA was sheared to 300-500bp and then biotinylated ligation junctions were recovered with streptavidin beads. standard Illumina library construction protocol, Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 8-12 cycles and library fragments of 400-600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeqX Ten following the manufacturer's protocols
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci – almost always bound by CTCF and cohesin – lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid. Overall design: we use in situ Hi-C, ChIP-Seq and PRO-Seq to probe nuclear architecture, the epigenome and transcription during cohesin loss and recovery
SAMPLE_TITLE: Rao-2017-HIC002
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Colorectal carcinoma"}, {"TAG": "cell line", "VALUE": "HCT-116-RAD21-mAC"}, {"TAG": "protocol", "VALUE": "in situ Hi-C"}, {"TAG": "tissue", "VALUE": "Colorectal carcinoma"}]
Pool_Member_@attributes_sample_title: Rao-2017-HIC002
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