EdU_BLM_20H_s
DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) at 37°C under a humidified atmosphere with 5% CO2. Cells were treated with 400µM mimosine (Sigma; M0253), 4h later cells were treated with 300 nM 4OHT (Sigma; H7904) for another 20h. EdU (5-ethynyl-2'-deoxyuridine from Invitrogen; 10µM final) was added 1h before cells were harvested. Genomic DNA was extracted with TEN/RNAse buffer (10mM Tris pH8, 100mM NaCl, 25mM EDTA, 0.5% SDS, 20µg/ml RNAseA) for 1h at 37°C before adding 20µl of Proteinase K (10mg/ml, Roche) for 2h at 56°C. Another 20µl of Proteinase K was added for O/N digestion. Genomic DNA was then purified with Phenol/chloroform (Sigma), precipitated with 70% ethanol (Sigma)/Na Acetate (0.3M, Invitrogen) and digested with MboI (125U per 100µl reaction volume) for 12h at 37°C followed by NlaIII (12.5U per 100µl reaction volume) for 4h at 37°C. Digested DNA was purified with Phenol/chloroform and precipitated as above. DNA was then biotinylated by click-it reaction according to the manufacturer protocol (Click-iT® Nascent RNA Capture Kit; Life technology) and precipitated with 70% EtOH (Sigma)/Na-acetate (0.3M, Invitrogen). Biotinylated DNA was pull-down using streptavidin beads according to manufacturer protocol (Click-iT® Nascent RNA Capture Kit; Life technology). DNA concentration on beads was measured using Qubit quantification and DNA on beads was resuspended in 50µl Elution buffer from Arima Hi-C Kit (BoxA).  The End Repair, dA-tailing and Adapter ligation steps were done using the NEB Next Ultra II DNA library Prep kit for Illumina as suggested by the manufacturer (Arima Hi-C Kit User guide).