GSM3775813: HATX_WT_input_Oct4_Rep1; Mus musculus; ChIP-Seq
Cells were then lysed (50mM HEPES-KOH, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 minutes at 4'C.  Nuclei were lysed (100 mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA) for 10 minutes at room temperature.  Chromatin was resuspended in sonication buffer (10 mM Tris pH 8.0, 1mM EDTA, 0.1% SDS) and sonicated using a Covaris Ultrasonicator to an average length of 220 bp. For all ChIPs, 5 million cells per replicate were incubated with 5 ug (Histone) or 7.5 ug (Oct4) antibody overnight at 4'C .  Antibody-bound chromatin was incubated with Protein G Dynabeads (Invitrogen, 10004D) for 4 hours at 4'C and eluted in Tris buffer (10mM Tris pH 8.0, 10mM EDTA, 1% SDS).  Crosslinks were reversed by incubation overnight at 65'C followed by treatment with 0.2 mg/mL proteinase K (Life Technologies, AM2548) and 0.2 mg/mL RNAse A (Qiagen).  DNA was purified using Qiagen Minelute Columns (Qiagen, 28006).  For library preparation, 4 ng ChIP DNA was incubated with transposase (Illumina, Fc-121-1030) for 10' at 55'C.  DNA was then amplified using Nextera barcoding adapters and sequenced on an Illumina Nextseq (2x75 bp reads).