GSM6481379: XD5_H3K4me3; Homo sapiens; OTHER
All cells required for one experiment were spun down for 3 min at 200 x g at RT, supernatant was removed and cell pellet was resuspended in 1.5 ml RT Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, EDTA-free protease inhibitor) by gently pipetting, spun down and washed twice more. After the last wash, supernatant was removed, the pellet was resuspended in 1 ml RT Wash Buffer. ConcavalinA-coated magnetic beads were activated by washing once in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) and added to the sample while gently vortexing at RT. Samples were incubated on a rotator for 10 min at RT. Sample was divided in aliquots (250k cells / ab) and placed on a magnet to remove the supernatant. 100 ul of antibody buffer + corresponding antibody (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.025% Digitonin, 4 mM EDTA and EDTA-free protease inhibitor) were pipetted along the sides of the tubes and pipetted up and down 10x. The following antibodies were used at a dilution 1:100: H3K27ac (ab4729, abcam), H3K4me1 (ab8895, abcam), MED1 (ab64965, abcam), H3K4me3 (07-473, Millipore), SOX9 (AB5535, Millipore) and IgG ( . The resuspended beads were rotated o/n at 4°C. The next day, tubes were quickly spun, put on a magnetic rack and supernatant was removed. 150 ul of Dig-wash buffer antibody (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.025% Digitonin, and EDTA-free protease inhibitor) were added and tubes mixed by inversion for four times. Supernatant was removed and 150 ul Protein-A-MNase (700 ng/ml) were added along the tube sites, mixed by pipetting and incubated for 1 h at 4°C on the rotator. Supernatant was removed, samples were washed four times with 150 ul Dig-wash buffer, resuspended in 100 ul Dig-wash buffer and stored in wet ice (0°C) to chill down. 2 ul of 100 mM CaCl2 were added and digestion was incubated for 30 min. Digestion reaction was stopped by adding 100 ul 2x STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 100 ug/ml RNAse A and 50 ug/ml Glycogen). After addition, samples were incubated at 37°C for 30 min for the release of soluble fragments to the supernatant, which was transferred to a new tube. Released DNA was purified by phenol-chloroform extraction. Sequencing libraries were produced using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645) as described by the manufacturer