EXPERIMENT_TITLE: GSM7512254: HCT116_HiC_Nut10h_BR2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were fixed in DMEM medium supplemented with 10% FBS and 2% methanol-free formaldehyde for 10 minutes rotating at room temperature. After quenching the formaldehyde with 0.125M glycine and washing the cells with 1X PBS, nuclei were extracted by lysing the cells in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA630 (SIGMA-ALDRICH #18896-50ML), 1× cOmplete EDTA-free protease inhibitor cocktail. Chromatin was digested overnight with HindIII enzyme and the cohesive restriction fragment ends were filled-in with dCTT, dTTP, dGTP and biotin-14-dATP(Invitrogen #19524-016) nucleotides. After blunt-end ligating the restriction fragment ends, the chromatin was decrosslinked by incubating overnight with proteinase K and purified by phenol:chloroform:isoamyl alcohol 25:24:1 (SIGMA-ALDRICH #P3803-100ML) extraction.Non-informative biotin at restriction fragment ends was removed by incubating the samples with dATP and T4 DNA polymerase (New England Biolabs#M0203S). After purifying the DNA again with a phenol:chloroform:isoamyl alcohol extraction, 10µg of the chromatin was sheared using a Covaris M220 focused ultrasonicator in 130µl cuvettes using the following parameters: 20% duty factor, 50 peak incident power, 200 cycles per burst, 65 seconds. The ends were end-repaired and dATP-tailed, followed by a biotin-pulldown using Dynabeads MyOne streptavidin C1 paramagnetic beads (Thermo Fisher #65001) to enrich for those DNA fragments which contain information of a chromatin loop. PE Illumina adapters(Supplementary Data 9) were ligated to the sample and the library was amplified for 8 cycles. Finally, the library was purified using a SPRI bead double-sided selection (0.4-1 volumes). Size and concentration of the finished Hi-C libraries was assessed by DNA ScreenTape Analysis (Agilent #5067-5582) on an Agilent 2200 Tapestation.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Activation of the p53 tumor suppressor triggers a transcriptional program to control cellular response to stress. However, the molecular mechanisms by which p53 controls gene transcription are not completely understood. Here, using a multi-omics integration framework, we uncover the critical role of spatio-temporal genome architecture in this process. We demonstrate that p53 drives direct and indirect changes in genome compartments, topologically associating domains and DNA loops within minutes of its activation, which escort the p53 transcriptional program along time. Focused on p53-bound enhancers, we report a core transcriptional program of 340 genes directly regulated by p53 over distance, most of these not previously identified. Finally, we showcase that p53 controls transcription of distal genes through newly formed and pre-existing enhancer-promoter loops in a cohesin dependent manner. Taken together, our findings demonstrate a previously unappreciated architectural role of p53 as regulator at distinct topological layers and provide a reliable set of new p53 direct target genes that may help future designs of p53-based cancer therapies. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27ac and H3K4me1 in HCT116 cells.
SAMPLE_TITLE: HCT116_HiC_Nut10h_BR2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HCT116"}, {"TAG": "cell line", "VALUE": "HCT116"}, {"TAG": "treatment", "VALUE": "Nutlin3a 10h"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HCT116_HiC_Nut10h_BR2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HCT116_HiC_Nut10h_BR2
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR25040644.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos6/sra-pub-zq-40/SRR025/25040/SRR25040644/SRR25040644.lite.1", "size": "10562954782", "date": "2023-07-03 07:32:30", "md5": "dddd64a5ca490551c757b59ac842f49c", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR25040644", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR25040644/SRR25040644", "size": "24719279392", "date": "2023-06-28 06:45:44", "md5": "7dbae7f4f878da057016ca01695cf240", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HCT116_HiC_Nut10h_BR2_2.fq.gz", "size": "15203186105", "date": "2023-06-28 06:29:57", "md5": "bce43ef078ec2420dadc1d36617f2c72", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HCT116_HiC_Nut10h_BR2_1.fq.gz", "size": "14662396276", "date": "2023-06-28 06:27:09", "md5": "cc0f36ca7850ebceeb257539a27b9b93", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]