GSM3669490: GS701 MM6 H3K27me3 ChIPseq rep2; Homo sapiens; ChIP-Seq
formaldehyde-fixed cells (1% formaldehyde, 10min RT) were lysed in Buffer B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0,3%SDS, 1x protease inhibitors -Roche) and sonicated in a microTUBE on a Covaris S220 until most of the DNA fragments were 200-500 base pairs long (settings: duty cycle 2%, peak incident power 105 Watts, cycles per burst 200, 25 min). After shearing lysates were centrifuged (10min 4°C 12000g) and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% DOC, 1x protease inhibitors-Roche). In parallel, Dynabeads (Protein A) were conjugated to the antibody (2ul, H3K27ac, diagenode, cat no: C15410174, lot no: A.7071-001P; 1.5ul, H3K27me3, Active Motif, cat no: 39161, lot no: 21518003) in PCR tubes by rotating 1.5 hour at 4°C (30rpm). Antibody-conjugated magnetic beads were added to the tube containing the chromatin and incubated 3h at 4°C (30rpm). Beads were washed (20rpm; 10min) four times with 500 ul Buffer A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with 500 ul Buffer C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2μl of RNase (10mg/ml) for 30 min at 37°C, 900 rpm, then 1h with 2 μl of Proteinase K (20mg/ml) at 55°C, 900 rpm, and finally overnitht at 65°C to revert formaldehyde crosslinking; beads were magnetized and supernatant was transferred to a new tube. Another 30 μl of elution buffer + 1μl of proteinase K were added to the beads and the tubes were incubated 1h with the same conditions than before; the eluates were combined. Finally DNA was purified with AMPure XP beads. Ultra II DNA Library prep kit for Illumina (NEB E7645S) was used for library preparation