GSM8195768: BLaER, MCC Promoter, 0h, rep 3; Homo sapiens; OTHER
Multiple aliquots of 15 M cells were crosslinked in 10 mL of culture medium with 2% formaldehyde (ThermoFischer, 28908). Next, the cells were permeabilized by digitonin (0.0025%; Sigma-Aldrich, D141). Digestion was performed with MNase (NEB, M0247) in low Ca2+ MNase buffer (10 mM Tris-HCl pH 7.5, 10 mM CaCl2) for 1 h at 37 °C. After confirming efficient digestion, samples were ligated and de-crosslinked. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, 69504). Before sonication, samples were size-selected to remove fragments corresponding to mono-nucleosomes using Mag-Bind TotalPure NGS beads (Omega Bio-Tek, M1378-01). MCC libraries were sheared using a Covaris S220 Focused-Ultrasonicator to a mean size of 200 bp (time: 280 s, duty factor: 10%, peak incident power: 175 W, cycles per burst: 200). Afterwards, the sonicated DNA fragments were indexed using 2 µg of DNA per indexing reaction. To increase library complexity, each reaction was indexed in duplicate in 7 PCR cycles using Herculase II polymerase (Agilent, 600677). The duplicate reactions were pooled and the libraires were enriched for viewpoints of interest in a double capture procedure, using ssDNA 5'-biotinylated 120 nt oligonucleotides. Briefly, 2 separate hybridization reactions were performed for each condition, using 2 µg of each sample as input. Captured DNA was pulled down using M-270 Streptavidin Dynabeads (Invitrogen, 65305), and amplified in 12 PCR cycles. After library quality assessment with a fragment analyzer, the libraries were sequenced on the Illumina sequencing platform with 300 cycles paired-end reads.