EXPERIMENT_TITLE: NextSeq 500 paired end sequencing; DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h. For ATM inhibition, cells were pretreated for 1 h with 20μM KU-55933 (Sigma, SML1109) and during subsequent 4OHT treatment. For ATR inhibition, cells were pretreated for 1 h with 2μM ETP-46464 (Sigma, SML1321) and during subsequent 4OHTsiRNA transfection were performed using the 4D Nucleofector X (kit SE cell line) according to the manufacturer's instructions and subsequent treatment(s) were performed 48 h later. siRNA sequences: CTRL: CAUGUCAUGUGUCACAUCU    SCC1: GGUGAAAAUGGCAUUACGG   WAPL: CGGACUACCCUUAGCACAA. For CRISPR/Cas9-mediated DSB induction, sgRNA (AsiSI site position: “CGCCGCGATCGCGGAATGGA” or Position further within the TAD=\"CRISPRsite\": “GGGCCAGTCGCGGCACTCGC”) were delivered in U2OS cells using the “nanoblades” technology which relies on direct cell transduction with a viral-derived particle containing the Cas9/sgRNA ribonucleoprotein (Mangeot et al, 2019). Cells were analyzed 24 h after transduction.    Hi-C experiments were performed in DIvA cells using the Arima Hi-C kit (Arima Genomics) according to the manufacturer's instructions. 1 million cells were used by condition. Briefly, cells were cross-linked with 2% formaldehyde for 10 min at RT, lysed and chromatin was digested with two different restriction enzymes included in the kit. Ends were filled-in in the presence of biotinylated nucleotides, followed by subsequent ligation. Ligated DNA was sonicated using the Covaris S220 to an average fragment size of 350 bp with the following parameters (Peak incident power: 140; Duty factor: 10%; Cycles per burst: 200; Treatment time: 70s). DNA was then subjected to a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter). Biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit).  Hi-C library was prepared on beads using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina (New England Biolabs) following instructions from the Arima Hi-C kit.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: DNA Double-Strand Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the tridimensional genome organization around DSBs and its role in DSB repair foci formation. We performed Hi-C experiments before and after DSB induction and upon ctrl or SCC1 depletion, 4C-seq experiments before and after DSB induction and upon cohesin (SCC1) depletion or ATM inhibition. We also performed ChIP-seq of pATM(S1981), CTCF, P-SMC3(S1083), MDC1 and a calibrated ChIP-seq of SCC1 with or without damage. ChIP-chip of SMC3 (S1083) and SMC1 (S966) were used to show the recruitment of these marks on DNA repair foci. ChIP-chip of gammaH2AX was realized upon SCC1 depletion and ChIP-seq of gammaH2AX was realized upon SCC1 or WAPL to show the role of the cohesin complex in gammaH2AX foci formation. ChIP-seq of gammaH2AX was realized upon ATM or ATR inhibition to show that ATM is the major kinase that phosphorylates H2AX at clean breaks in human cells.
SAMPLE_TITLE: HiC_siCTRL_mOHT
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "ENA first public", "VALUE": "2021-01-29"}, {"TAG": "ENA last update", "VALUE": "2021-01-06"}, {"TAG": "External Id", "VALUE": "SAMEA7767563"}, {"TAG": "INSDC center alias", "VALUE": "LBCMCP, UMR5088, CBI"}, {"TAG": "INSDC center name", "VALUE": "LBCMCP, UMR5088, CBI"}, {"TAG": "INSDC first public", "VALUE": "2021-01-29T04:16:26Z"}, {"TAG": "INSDC last update", "VALUE": "2021-01-06T17:31:34Z"}, {"TAG": "INSDC status", "VALUE": "public"}, {"TAG": "Submitter Id", "VALUE": "E-MTAB-8851_2:HiC_siCTRL_mOHT"}, {"TAG": "broker name", "VALUE": "ArrayExpress"}, {"TAG": "cell line", "VALUE": "U2OS"}, {"TAG": "common name", "VALUE": "human"}, {"TAG": "genotype", "VALUE": "AsiSI-ER"}, {"TAG": "sample name", "VALUE": "E-MTAB-8851_2:HiC_siCTRL_mOHT"}]
Pool_Member_@attributes_sample_title: HiC_siCTRL_mOHT
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HiC_siCTRL_mOHT
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