title: GSM3213969: B6WT_72h_DSG_Tet2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were centrifugation at 250 x g for 5 mins and cell pellets were resuspended in 37°C PBS with 2mM disuccinimidyl glutarate (ThermoFisher) to crosslink proteins for 30 mins at room temperature. Formaldehyde was added to a final concentration of 1% and the cells were incubated at room temperature for 10 mins. Fixation was quenched with 125 mM glycine and cells were washed twice with ice cold PBS. Cells were pelleted, snap-froze with liquid nitrogen. To isolate nuclei for sonication, cell pellets were thawed on ice and lysed with lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) for 10 mins at 4°C with rotation, washed once with washing buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and twice with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS). Nuclei were resuspended in 1mL shearing buffer and sonicated with Covaris E220 using 1 mL milliTUBE (Covaris, Woburn, MA) for 18-20 minutes (Duty Cycle 5%, intensity 140 Watts, cycles per burst 200). After sonication, insoluble debris was removed by centrifugation at 20,000 x g. Buffer for chromatin was adjusted with 1 volume of 2x conversion buffer (10 mM Tris-HCl pH 7.5, 280 mM NaCl, 1 mM EDTA, 1mM EGTA, 0.2% sodium deoxycholate, 0.2% Triton-X100, 1% Halt protease inhibitors with 0.1% SDS). Chromatin was pre-cleared with washed protein A dynabeads  (ThermoFisher) for 2 hours, incubated with rabbit polyclonal anti-Tet2 (ab124297;  Abcam) and protein A dynabeads overnight (all procedures were at 4°C with rotation). Bead-bound chromatin was washed twice with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS), once with high salt wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was eluted from beads with elution buffer (100 mM NaHCO3, 1% SDS, 1 mg/mL RNaseA; Qiagen) twice for 30 mins each at 37°C with constant shaking. NaCl and proteinase K (Ambion) were added to the eluted chromatin at concentrations of 250 mM and 0.5 mg/mL, respectively, and de-crosslinked at 65°C overnight with constant shaking. DNA was purified with Zymo ChIP DNA Clean & Concentrator-Capped Column (Zymo Research, Irvine, CA). Library was prepared with NEB Ultra II library prep kit (NEB) following manufacture's instruction. ChIP-seq, 50bp single end with HiSeq2500
attributes.tag: GEO Accession
attributes.value: GSM3213969