GSM6922671: MCC_replicate_2; Homo sapiens; OTHER
Cells fixed with 2% formaldehyde and lysed on ice Briefly, 10^7 SEM cells were fixed with 2% formaldehyde for 10 min, then quenched with glycine and washed with PBS. Cells were permeabilized with 0.005% digitonin (Sigma Aldrich) for 15 min, then snap frozen. Thawed cells were pelleted and resuspended in reduced-calcium MNase buffer (10 mM Tris-HCl pH7.5, 1 mM CaCl2, then split into three aliquots. Cells were treated with a titration of different MNase (NEB) concentrations (typically 10-30 Kunitz U in an 800 µl volume) for 1h at 37 ˚C in a Thermomixer (Eppendorf) at 550 rpm. The reaction was stopped with addition of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) to 5 mM and 200 µl was removed to assess the extent of digestion. The remaining cells were pelleted, washed with PBS/EGTA, then processed for end repair, phosphorylation and ligation. Cell pellets were resuspended in DNA ligase buffer, supplemented with 400 µM dNTPs and 5 mM EDTA, before addition of DNA Polymerase I large (Klenow) fragment (NEB) to 100 U/µl, T4 polynucleotide kinase (NEB) to 200 U/µl and T4 DNA ligase (Thermo Scientific) to 300 U/µl. The reaction was incubated for 2h at 37 ˚C, then for 8h at 20 ˚C, at 550 rpm, then cooled to 4 ˚C overnight. Both the digested and ligated chromatin was decrosslinked at 65 ˚C in the presence of proteinase K, then DNA was purified using the Qiagen DNeasy Blood and Tissue Kit. Digestion and ligation efficiencies were assessed by TapeStation (Agilent D1000). 2 ug DNA was adapter ligated using a strategy based on sonication combined with the NEBNext Ultra II DNA Library Prep kit. The quality of the libraries was assessed using the Agilent ScreenTape system. Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments. 700 ng of the adapter ligated libraries was hybridized with the biotinylated capture oligonucleotides, using the HyperCapture Target Enrichment Kit (Roche). The quality of the resulting captured library was assessed using the Agilent ScreenTape system.