title: GSM8657304: chALL1 Sub-library 3; Homo sapiens; OTHER
library_construction_protocol: MCC protocol Briefly, 5x106 chALL1 blast 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 pH 7.5, 1 mM CaCl2), then split into 3 aliquots. Cells were treated with a titration of different micrococcal nuclease (NEB) concentrations (typically 10-30 Kunitz U in an 800 µl volume) for 1 h at 37 °C in a Thermomixer (Eppendorf) at 550 rpm. The reaction was stopped with the 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 2 h at 37 °C, then for 8 h at 20 °C, at 550 rpm, then cooled to 4 °C overnight. Both the digested and ligated chromatin were 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). Library preparation, indexing and capture were performed as described for NG Capture-C.