EXPERIMENT_TITLE: GSM5478476: CkoC44-NT-R1-T1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 5-10M formaldehyde cross-linked cells were lysed and digested overnight at 37°C with 400 units of DpnII (NEB). Next, the DNA ends were marked with biotin-14-dATP (Thermo Fisher Scientific #19524016) for 4 hours at 23°C and ligated for 4 hours at 16°C. To remove proteins and reverse crosslink, DNA was treated with proteinase K overnight at 65°C. DNA was then purified using phenol:chloroform and biotin was removed from unligated ends. Ligation products were fragmented by sonication to an average size of 200bp and size selected with Ampure XP beads (Beckman Coulter, # A63880) to fragments ranging from 100 to 350 bp. End repair was performed on the DNA fragments to obtain blunt ends and biotinylated ligation products were purified using streptavidin beads (Thermo Fisher Scientific #65001). dA-tailing was then performed and the TrueSeq DNA LT kits Set A (Illumina, #15041757) was used for adapter ligation and PCR amplification. PCR primers were removed using Ampure XP beads. The Hi-C libraries were sequenced using 50 bp paired end reads on Illumina HiSeq4000.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Cohesin-mediated loop extrusion folds interphase chromosomes at the ten to hundreds kilobases scale. This process produces structural features such as loops and topologically associating domains. We identify three types of cis-elements that define the chromatin folding landscape generated by loop extrusion. First, CTCF sites form boundaries by stalling extruding cohesin, as shown before. Second, transcription termination sites form boundaries by acting as cohesin unloading sites. RNA polymerase II contributes to boundary formation at transcription termination sites. Third, transcription start sites form boundaries that are mostly independent of cohesin, but are sites where cohesin can pause. Together with cohesin loading at enhancers, and possibly other cis-elements, these loci create a dynamic pattern of cohesin traffic along the genome that guides enhancer-promoter interactions. Disturbing this traffic pattern, by removing CTCF barriers, makes cells sensitive to deletion of genes involved in transcription initiation, such as the SAGA and TFIID complexes, and RNA processing such DEAD-Box RNA helicases. In the absence of CTCF, several of these factors fail to be efficiently recruited to active promoters. We propose that the complex pattern of cohesin movement along chromatin contributes to appropriate promoter-enhancer interactions and localization of transcription and RNA processing factors to active genes. Overall design: We engineered a HAP1-derived human cell line in which CTCF or RNApolII (RPB1) could efficiently be removed using an auxin-inducible degron system. We depleted the DDX55 and TAF5L proteins in HAP1-CTCFdegron-TIR1 cells in two ways: siRNA and knock-outs. We performed Hi-C on control cells, cells depleted for CTCF or RPB1 or DDX55 or TAF5L or combinations of CTCF depletions with DDX55 or TAF5L depletion.
SAMPLE_TITLE: CkoC44-NT-R1-T1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HAP1 cells"}, {"TAG": "cell line", "VALUE": "HAP1"}, {"TAG": "treatment", "VALUE": "non-treated"}, {"TAG": "enzyme", "VALUE": "DpnII"}, {"TAG": "genotype", "VALUE": "endogenous CTCF KO, CTCF-AID"}]
Pool_Member_@attributes_sample_title: CkoC44-NT-R1-T1
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