GSM2792782: Input_DNA_day3_ePOUSKM; Mus musculus; ChIP-Seq
Cells were resuspended in PBS at a density of 5 x 10^5/ml and then chemically crosslinked by the addition of 1% formaldehyde for 10 min at room temperature with gentle rotation. Crosslinking was stopped by the addition of glycine to a final concentration of 125 mM for 5 min at room temperature. Cells were washed twice with 10 ml cold PBS and lysed in 10 ml lysis Buffer 1 (150 mM NaCl, 0.5% NP40; 1 mM EDTA, 10% glycerol, 0.25% triton-X100) freshly added complete proteinase inhibitor cocktail in 15 ml tubes at 4 °C for 10 min with gentle rocking. The lysate was centrifuged for 10 min at 1350 x g at 4°C. The supernatant was discarded and the pellet was resuspended in 10 ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitor) incubated at 4°C for 10min. The procedure was repeated using 10 ml lysis buffer 3 (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, protease inhibitor) and 10 min rocking at RT. Nuclei were collected by centrifugation and washed twice with sonication buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, protease inhibitor) and finally suspended in sonication buffer. Samples were sonicated using a Covaris S220 machine with settings to obtain DNA fragments with a length from 300 to 400 bp using microTUBEs (130 μl) for 5 cycles of 30 sec ON and 60 sec OFF using default settings recommended for microTUBEs. 1/10th volume of 10% Triton-X100 was added and the lysate was centrifuged at high speed (20,000 x g for 10 min at 4°C). 1/10th of the supernatant was stored as input control. ChIP was performed using the IP-Star compacted automated system (Diagenode) using 6 μg ChIP-grade Oct4 (Santa Cruz; # Sc-8628X) and Sox2 (Santa Cruz; #sc-17320X) antibodies with 80 μl protein G Dynabeads with IPure 8 samples 200 μl protocol. The coupling time for beads and antibody was 5 hours followed by incubation with lysate for 15 hours at 4°C. Automated wash steps of 10 minutes at 4°C were performed in the following order: low salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS 1% TritonX-100, 2 mM EDTA, 150 mM NaCl), high salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS 1% TritonX-100, 2 mM EDTA, 500 mM NaCl), LiCl buffer (500 mM LiCl, 50 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate), TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) with final elution in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS buffer. The eluted chromatin and input samples were reverse‐crosslinked by overnight incubation at 65°C. Samples were treated with RNaseA (0.2 mg/ml final concentration) at 37°C for 2h followed by proteinase K (0.2 g/ml final conc.) at 55°C for 3 hours. DNA was extracted by phenol:chloroform:isoamyl alcohol (25:24:1) and phase separated using heavy phaselock tubes by centrifugation at 10000 rpm for 10 min at RT and precipitated with 100% ethanol with 20μg/μl glycogen as co-precipitant. The precipitate was washed with 80% ethanol and finally eluted in 20ul water. DNA concentration was determined using Qubit dsDNA HS assay kit (Thermo Fisher Scientific; #Q32851). The libraries were generated using the Trueseq Nano DNA Lib prep kit (Illumina)
GEO Accession
GSM2792782