GSM7466901: HUWE1-WT, shCtrl, DSBseq, rep2; Homo sapiens; OTHER
Double strand break sequencing was performed based on the DSBCapture protocol (Lensing et al., 2016). In brief, 5 million cells were fixed with 90% methanol. Cells were permeabilized in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% Triton-X100) for 10 min on ice and bound to Con-A Sepharose (Sigma) in binding buffer (20 mM HEPES, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). DNA ends were repaired using Klenow fragment (NEB) in NEB buffer 2 for 30 min at 25°C followed by A-tailing in dA-tailing buffer (NEB) with Klenow (3'→5' exo-) for 30 min at 37°C. Biotinylated NEB adaptors were ligated to A-tailed DSBs with T4 DNA ligase (NEB) in the corresponding buffer overnight at 16°C. Cells were lysed in 10 mM Tris, pH 8.5, 5 mM EDTA, 0.5% SDS, 250 mM NaCl, 1.2 U Proteinase K (Thermo) at 50°C overnight followed by sonication for 5 min on ice and DNA purification using phenol/chloroform/isoamyl and ethanol. Biotinylated DNA was captured on streptavidin C1 beads (NEB) and washed with 10 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.02 % TritonX100. Sequencing libraries were prepared using NEB Ultra II DNA library Prep kit according to the manufacturer's instructions.