EXPERIMENT_TITLE: GSM7980128: HIC_Pdef_DSB_rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Chipseq: Cultured cells were fixed with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 125 mM glycine. Twenty million fixed cells were washed with PBS, snap-frozen and stored at -80°C until further processing. Before use, the cells were resuspended in 1 ml of RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 and freshly added Complete Mini EDTA free proteinase inhibitor. Sonication was performed using Branson sonicator at amplitude 35%, 12 cycles of 20 sec sonication followed by 30 sec of pause or using Covaris ME220 sonicator at duty factor 20%, peak incident power 75, cycle/burst 1000 for 6 min at 4°C. The antibody-protein solution was then incubated with 40 ml of Dynabeads Protein A for 40 min at room temperature under agitation. Antibody-bound beads were washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with LiCl buffer (0.25M LiCl, 0.5% NP-40, 0.5% NaDOC), once with TE pH 8.0 containing 0.2% Triton X-100, and once with TE pH 8.0. Crosslinks were reversed by incubating the beads at 65°C for 4 hours in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified by ChIP DNA clean and concentrator column. HiC: Cultured cells were fixed with 1% formaldehyde and the reaction was quenched with 200 mM glycine. Cells were then lysed and digested with the MboI restriction enzyme. DNA ends were then marked with biotin-14-dATP and ligated by proximity. After reverse crosslinking, DNA was sheered to an average size of 200-400 bp. This DNA was then pulled down using MyOne Streptavidin T1 beads and washed. Chipseq: Ovation Ultralow Library System V2 kit , Hi-C library were amplified directly from of the beads with 4-6 cycles of PCR, using Illumina primers (Bioo Scientific) and Q5 Hi-Fidelity DNA polymerase
STUDY_DESCRIPTOR_STUDY_ABSTRACT: we describe and clarify several mechanisms whereby ?Cohesin contributes to DNA repair. Upon acute Cohesin depletion, we observed delayed DNA repair and increased frequency of translocations. These translocations were directly correlated to contact frequency between damaged domains. We found that phosphorylation of the Cohesin subunit SMC1A is required to direct specific Cohesin recruitment at DSBs, without affecting its distribution in the undamaged genome. Moreover, Cohesin depletion caused a significant increase in translocations in the 53BP1 deficient background, indicating that Cohesin functions to suppresses 53BP1 independent end joining mechanisms. Our work sheds light on a previously unappreciated mechanisms whereby gCohesin is loaded at DSBs and actively extrudes loops between the break and nearest natural TAD boundary. This process modulates the 3D proximity of broken ends to accelerate repair speeds and prevent long range end joining events during NHEJ. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Rad21, Nipbl and Mre11 in HCT116 cells in different genotypic background w/wo DSB and HiC analysis w/wo auxin in DSB.
SAMPLE_TITLE: HIC_Pdef_DSB_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HCT116"}, {"TAG": "cell line", "VALUE": "HCT116"}, {"TAG": "cell type", "VALUE": "Human colorectal carcinoma cell"}, {"TAG": "genotype", "VALUE": "Rad21_AIDdegron, SMC1AS957AHz/S966AHz"}, {"TAG": "treatment", "VALUE": "DSB(4OHT)"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HIC_Pdef_DSB_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HIC_Pdef_DSB_rep2
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