GSM3381542: Sox2_O4SK_d5; Mus musculus; ChIP-Seq
Reprogramming cells (7.5-10 x 10e6 cells per sample were collected by trypsinization at day 1, 5 and 7 days post transduction and washed twice with PBS (Gibco). Cells were resuspended in PBS at a density of 5 x 105/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 10 min. 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 150 to 500 bp using microTUBEs (130 μL) for 5 cycles of 30 sec ON and 60 sec OFF using default settings recommended for microTUBEs. 10% Triton-X100 was added and the lysate was centrifuged at high speed (20000 x g for 10 min at 4°C). 1/10 of the supernatant was stored as input control. ChIP was performed in an automated fashion using the IP-Star compacted automated system (Diagenode) using 6 ug ChIP-grade antibodies (Oct4 or Oct6) and 50 ul protein G Dynabeads (Invitrogen). 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 ug/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 phase-lock tubes (TianGen) by centrifugation at 10000 rpm for 10 min at RT and precipitated with 100% ethanol with 20 μg/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. The libraries were generated using the Trueseq Nano DNA kit (Illumina)
GEO
GSM3381542
GEO Accession
GSM3381542