GSM9065069: HAP1 TAF5L KO anti-KAT2A Cut&Run rep2; Homo sapiens; OTHER
For each sample, nuclei from 250,000 cells were isolated in 100 µl nuclear extraction (NE) buffer (20 mM HEPES (Thermo Fisher, 15630-056), 10 mM KCl (Thermo Scientific, AM960-G), 0.1 % Triton X-100 (Sigma, 93443-100ML), 20 % Glycerol (Fisher Scientific, 10692372), 0.5 mM Spermidine (Sigma, S2626-1G) supplemented with Roche cOmplete EDTA-free Protease Inhibitors (Sigma, 11873580001) for 10 min on ice and centrifuged for 3 min at 600 g at room temperature Samples were resuspended in 100 µl NE buffer and bound to 10 µl of activated ConA beads for 10 min at room temperature. Next, beads were collected with a magnetic rack and resuspended in 50 µl Antibody Buffer (20 mM HEPES, 150 mM NaCl (Fisher Scientific, BP358-1), 0.5 mM Spermidine, 0.02 % Digitonin (Millipore, D141-100MG), 2 mM EDTA supplemented with Roche Complete EDTA-free Protease Inhibitors) supplemented with 1 µl of antibody. Nuclei were incubated overnight on a nutator at 4C. After two washes with 250 µl Digitonin buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, 0.02 % Digitonin supplemented with Roche Complete EDTA-free Proteasome Inhibitor), nuclei were resuspended in 50 µl Digitonin buffer supplemented with 700 ng/µl pAG-Mnase (gift of Joris Van der Veeken, IMP, Vienna), and incubated for 30 min at room temperature. Excess pAG-Mnase was removed with two washes with Digitonin buffer. Finally, nuclei were resuspended in 50 µl Digitonin buffer and pAG-Mnase was activated with 1 µl 100 mM CaCl2 for 1.5 h on ice. The reaction was quenched with 33 µl Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA (VWR, J60767AD), 50 µg/mL RNAse A, 50 µg/mL Glycogen (Merck, 10901393001). Cleaved DNA was allowed to diffuse out by incubating the tubes at 37ºC for 10 min. After separation using a magnetic rack, DNA was isolated using the Monarch DNA Cleanup Kit (NEB, T1030) and eluted in 15 µl Elution Buffer. Next-generation sequencing libraries were prepared from 2 ng DNA using the NEBaNext Ultra II DNA Library prep Kit (NEB, E7645L), pooled and sequenced (PE150) at Azenta, at a depth of 5-10M reads per library.