GSM5448559: ChIP_H3.3_T181_7J; Mus musculus; ChIP-Seq
For the standard ChIP protocol 25 million ES cells were cross-linked in 7-8mL pre-tempered ES medium containing 1% formaldehyde (Pierce 28906) for 10min at 22°C. Fixation was stopped by addition of 0.125M final concentration of glycine, followed by two washing steps with RT PBS containing 10% Serum. Fixed cells were resuspended in 10mL ice cold buffer LB1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.50% NP-40, 0.25% Triton X-100, 1x Roche complete protease inhibitors (Roche, 04693159001)), rocked at 4°C for 10min and after centrifugation resuspended in 10mL ice cold buffer LB2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1x protease inhibitors), rocked at 4°C for 5min and centrifuged again. The pelleted nuclei were resuspended in 1mL ice cold shearing buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.1% SDS, 1x protease inhibitors). Buffer compositions for LB1, LB2 and shearing buffer are based on Boyer et al., 2005. Exactly 1mL was transferred to a 1mL milliTUBE with AFA fiber (Covaris 520130) and sonicated using a Covaris S220 device for a time of 15-20min and the following settings: Peak power 140 Watt, Duty factor 20%, Cycles per burst 200, Temperature of bath 4°C. The sheared samples were added to a 1.5mL tube with 110µL of 10% Triton X-100 (final concentration 1%) and centrifuged at 14000 rpm for 15 minutes at 4°C. The soluble chromatin containing supernatant was divided in 110µL aliquots (equivalent to 2-2.5Mio cells) and for IP diluted with 890µL complete 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). Per IP 30µL of Protein G Dynabeads (Invitrogen, 10004D) were used. For FLAG ChIP-seq experiments 1-2µL of anti -FLAG M2 antibody (Sigma-Aldrich, F3165) were used per IP and 4-5 IPs (equivalent to 10-12 million cells) were pooled to obtain enough material for library preparation. For H3K9me3 ChIP-seq experiments 1µL of anti-H3K9me3 (Active Motif, 39161) was used per IP. Beads were incubated with the corresponding antibody in complete Buffer A for 1.5h prior to IP, followed by two washes with Buffer A (without protease inhibitors). Diluted Chromatin was added to the pre-bound beads and incubated for 4h on a rotating wheel with 30 rpm at 4°C. After IP beads were washed 5 times with Buffer A and 1 time with Buffer C (10 mM Tris-HCl pH 8.0, 10 mM EDTA). Beads were resuspended in 100µL elution buffer (10 mM Tris HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS), RNA was degraded by 2μl RNase A (10mg/mL) for 30min at 37°C, proteins were digested with 2μl Proteinase K (10 mg/mL) at 55°C for 1h and crosslink reversal of immunoprecipitated DNA was carried out overnight at 65°C. DNA was purified using the Agencourt AMPure XP beads (Beckman Coulter, A63882). The Low input protocol is adapted from Dahl and Collas, 2008. It used 2Mio cells lysed in 100µL Buffer-B (50mM Tris-HCl, pH 8.0, 10mM EDTA, 1%SDS, 1x protease inhibitors) and sonicated in a microtube (Covaris; 520045) using the Covaris S220 device with the settings: Peak power 105 Watt, duty factor 2%, cycles per burst 200, Temperature of bath 4°C. The supernatant after centrifugation was diluted with 900µl complete Buffer A and 150µL (corresponding to approximately 300.000 cells) were used per IP with 10µL Protein G Dynabeads and 1µL of anti-H3K9me3 antibody as described above. Ultra II DNA Library prep kit for Illumina (NEB E7645S)