NextSeq 500 sequencing; DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
DNA double-strand breaks and 3D chromatin conformation (Hi-C, 4C-seq, ChIP-seq)
4Cseq_siSCC1pOHT_DSB1_rep2_s
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.    The 4C-seq experiments were realized as in Matelot and Noordermeer (2016) with minor modifications. Briefly, 15 million DIvA cells were cross-linked with 2% formaldehyde for 10 min at RT and the reaction was stopped using glycine (0,125M final) for 5min on ice. Cells were lysed, permeabilized with A50µL SDS 0,5% and digested with MboI (New England Biolabs). Three rounds of 4 h of digestion with MboI were necessary. Digested DNA was then ligated with a T4 DNA ligase (HC) (Promega), purified and digested with NlaIII overnight (New England Biolabs). After a second ligation step, DNA was purified before proceeding to the library preparation. For DNA purification steps, AMPure XP beads (Beckman Coulter) were used diluted at 1:10 in 20% PEG solution (PEG 8000 (Sigma) 20%, 2.5M NaCl, Tween 20 20%, Tris pH8 10mM, EDTA 1mM).  For 4C-seq library preparation, 800ng-900ng of 4C-seq template was amplified using 16 individual PCR reactions with inverse primers (PAGE-purified) including the Illumina adapter sequences and a unique index for each condition. Libraries were purified with the QIAquick PCR Purification Kit (Qiagen). The primer sequences used to amplify each viewpoint were:  Ctrl: FW:ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTCAGGTTATCATCCCAA REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACCTTCGCTGTACCTTTG DSB1: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCTGGCAACTTATGAATCAGGA REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGTCAAAAGCCAAGGGGACA DSB2: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCTTACGATTATTTGTGAATTTTG REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGCTAATTCTGAGTTACATACATT DSB3: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGATTACGTAGAAGGGTGCC REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGGCAAATGATAACCCTGT DSB4: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTTATACTAAGATGTCAGTTCCT REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACGCACCTGGTTTAGATT 470kbleft: FW:AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAAGGAAGAAGCAGGCATTCA REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTGAAATGAGTACTCTGCCATCCA CRISPRsite: FW:ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTAAGCACCCTCCTCCTAG REV:CAAGCAGAAGACGGCATACGAGAT(index6bp)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCTTTACACCTCAAAACCT.