GSM7817920: A549-ACE2_TNFa_induced_p65; Homo sapiens; OTHER
Cells were pelleted after scraping and resuspended in wash buffer (HEPES 20mM, NaCl 150mM, Spermidine 0.5mM) at 107 cells/ml. At room temperature, per sample, 10 μl of Concanavalin A beads slurry washed in bead activation buffer (HEPES 20mM, KCl 10mM, CaCl2 1mM, MnCl2 1mM) was added per 106 cells in the total volume of 1 ml of wash buffer and incubated for 10 min with rotation at 15–20 rpm. Antibodies against H3K27ac (Millipore, MABE647) were diluted 50 times, p65 (Cell Signaling, D14E12)  diluted 100 times and IgG (Invitrogen, 31235) 500 times in the buffer (wash buffer with 0.1% digitonin, 2 mM EDTA) and 50 μl of diluted antibody was added to the cells fixed on beads and incubated for 1h. Afterward, the beads were washed twice with 250 µl of digitonin buffer (wash buffer with 0.1% digitonin),  resuspended in 52 µl digitonin buffer containing 35ng of  pAG-MNase (produced at Institut Curie platform) , and incubated 10 min with agitation. Beads were washed twice in 250 µl of ice-cold digitonin buffer, resuspended in 100 µl of ice-cold digitonin buffer, kept on ice for 5 min before adding 2 µl of 100 mM CaCl₂ and incubating for 30 min on ice. The reaction was stopped by addition of 100 µl of stop buffer (NaCl 340mM, EDTA 20mM, EGTA 4mM, RNase A 50 μg/mL, glycogen 50 μg/mL and 0.1% digitonin) and incubated for 10 min at 37°C. Bead suspension was centrifuged at 16k rcf at 4°C for 5 min and beads were recovered for DNA fragments purification with NEB Monarch PCR & DNA purification kit, following the protocol enriching for short DNA fragments.