EXPERIMENT_TITLE: GSM3456999: BRCA1_MUT2 Hi-C; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Harvested cells were washed once with PBS, fixed with 2% formaldehyde for 10 min in PBS at room temperature, quenched with 0.2M glycine for 5 min. After washing once with PBS, pelleted cells were flash frozen and stored at -80°C for further processing. Nuclei were isolated with lysis buffer (10mM Tris-HCl (pH 8.0), 10mM NaCl, 0.2% IGEPAL CA630) with incubation on ice for 5 min, washed once same lysis buffer. Pelleted nuclei were permeabilized with 0.5% SDS for 10 min at 62°C, quenched with Triton X-100 for 15 min at 37 °C. Chromatin was digested with a 4 cutter restriction enzyme (MboI, 100U, NEB) overnight at 37°C with mixing. After inactivating digestion at 62°C for 20 min, the 5' overhangs were filled in with biotinylated-14-dATP (Life Tech) and Klenow (40U, NEB) for 90 min at 37°C with mixing. Biotinylated ends were ligated with T4 DNA ligase (2000U, NEB) for 4 hours at room temperature with mixing. DNA was reverse crosslinked with Proteinase K (400mg, NEB) and 1% SDS for 30 min at 55 °C, followed by an overnight incubation at 68°C with NaCl. DNA was purified with ethanol precipitation, and sheared on an ultrasonicator (Covaris S220; duty cycle: 10; intensity: 4; cycles/burst: 200; duration: 55s; number of cycles: 1) to a fragment range of 300-700 bp. Double size selection was performed using SPRI beads (Beckman Coulter). Biotinylated fragments were enriched by pulldown with Dynabeads MyOne T1 Streptavidin beads (Life Technologies), followed by library preparation (Quick Ligation Kit, NEB). Final library was amplified by PCR (Phusion DNA Polymerase, Thermo). Libraries were sequenced on HiSeq4000 Paired-End for 100bp (Illumina).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: We investigated BRCA1-deficient ovarian cancer to understand why homologous recombination deficiency (HRD) is associated with tumor T-cell inflammation. In humans and mice, BRCA1 deficiency led to increased cytoplasmic translocation of nuclear DNA, increased DNA sensing, induction of proinflammatory cytokines and T-cell recruiting chemokines, and increased tumor CD8+ T-cell infiltration. This cascade was mediated by STING and phosphorylation of TBK1, IRF3 and STAT1. BRCA1 loss activated a transcriptional reprogramming of tumor cells, leading to overexpression of the DNA sensing pathway and hyper-responsiveness to cytoplasmic DNA. Genetic alterations of the DNA sensing and interferon pathways modulated the impact of HRD on T-cell inflammation. PARP inhibitor olaparib exacerbated cytoplasmic DNA and the cell-autonomous inflammatory activation of BRCA1-deficient cancers, and rendered them susceptible to PD-1/CTLA-4 blockade. We conclude that HRD and DNA sensing drives the immunogenicity of ovarian carcinomas and predisposes them to immune vulnerability under immune checkpoint blockade. Overall design: The human ovarian cancer cell line UWB1.289,which harbors a deleterious mutation in BRCA1 exon11 and LOH, was profiled its chromatin structure by HiC. Same experimental procedure was followed for UWB1.289 BRCA1 wt reconstituted isogenic pair.
SAMPLE_TITLE: BRCA1_MUT2 Hi-C
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "UWB1.289 with mutation in BRCA1 - replicate 2"}, {"TAG": "cell line", "VALUE": "UWB1.289"}, {"TAG": "genotype", "VALUE": "BRCA1 2594delC"}]
Pool_Member_@attributes_sample_title: BRCA1_MUT2 Hi-C
RUN_SET_RUN_Pool_Member_@attributes_sample_title: BRCA1_MUT2 Hi-C
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR8157465.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos4/sra-pub-zq-1/SRR008/8157/SRR8157465/SRR8157465.lite.1", "size": "19944464944", "date": "2022-07-28 12:09:58", "md5": "e9051bb588e157613ff19a2f486ca8a7", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR8157465", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR8157465/SRR8157465", "size": "34856697984", "date": "2018-11-05 21:16:31", "md5": "67dd09c17d3fdd8ecc60168d16206faf", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}]