EXPERIMENT_TITLE: GSM3139148: Vian-2018-HIC209; 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, NextSeq 500, or HiSeq 2500 following the manufacturer's protocols
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes" where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development. Overall design: ChIP-seq, ChIA-PET, insitu Hi-C, 4C, localHiC, ChromRNAseq, and/or RNAseq from mouse stem cells, activated B cells, CH12 cell line, and plasmacytomas
SAMPLE_TITLE: Vian-2018-HIC209
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Colorectal carcinoma"}, {"TAG": "genotype", "VALUE": "Rad21-mAC"}, {"TAG": "cell line", "VALUE": "HCT-116-RAD21-mAC"}, {"TAG": "protocol", "VALUE": "in situ Hi-C"}, {"TAG": "treatment", "VALUE": "6 hours 500uM IAA, 60 min withdraw; 6 hours 300nM flavopiridol"}]
Pool_Member_@attributes_sample_title: Vian-2018-HIC209
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