GSM8489035: SK-N-SH-A Jun rep2; Homo sapiens; OTHER
250k cells per sample were dissociated, collected and washed once with DPBS. For nuclear isolation, cell pellets, after centrifugation at 400 g for 3 min, were resuspended in 100 µl nuclear extraction buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 % Triton X-100, 20 % glycerol, 1 mM MnCl2, 0.5 mM Spermidine, 1x cOmplete protease inhibitor cocktail (Roche)) and lysed at 4 °C for 10 min. Then, nuclei were pelleted at 400 g for 3 min and resuspended in 100 µl nuclear extraction buffer. 10 µl of Concanavalin A beads (Polysciences) were washed twice with 100 µl bead activation buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and resuspended in 10 µl of bead activation buffer.  Following, 100 µl nuclei were immobilized by incubation with 10 µl activated Concanavalin A beads (Polysciences) for 10 min at RT. Upon incubation of the nuclei-bead complex, the supernatant was removed and the beads were resuspended in 50 µl antibody buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x cOmplete protease inhibitor cocktail (Roche), 0.01 % digitonin, 2 mM EDTA) containing the antibodies and incubated at 4 °C while nutating overnight. Antibodies were used diluted as follows: 1:50 anti-YAP1 (Cell Signaling Technology, D8H1X, #14074), 1:25 anti-WWTR1 (Sigma-Aldrich, #HPA00741), 1:50 anti-JUN (Cell Signaling Technology, 60A8, #9165), 1:50 anti-H3K27Ac (Active Motif, #39133), 1:50 anti-H3K4me1 (Diagenode, #C15410194), 1:50 normal IgG (Cell Signaling Technology, #2729). Afterwards, the beads were washed twice with digitonin buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x cOmplete protease inhibitor cocktail (Roche), 0.01 % digitonin), and incubated with 700 ng/ml protein A/G-conjugated micrococcal nuclease (pAG-MNAse - kind gift from Joris van der Veeken, IMP) in 50 µl digitonin buffer at RT for 30 min. After two washes with digitonin buffer, the beads were resuspended in 50 µl digitonin buffer and digestion started by addition of 1 µl 100 mM CaCl2 and incubation at 4 °C for 90 min. The reaction was stopped by addition of 33 µl stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 50 µg/mL glycogen), followed by incubation at 37 °C for 10 min to release cleaved chromatin. DNA was extracted from the supernatant using Monarch DNA and PCR cleanup kit (New England Biolabs) according to manufacturer's instructions. 2 ng of DNA were used for library preparation using the NEBNext ultra II DNA library Prep kit (New England Biolabs).