GSM5832269: Case_LCL_input; Homo sapiens; ChIP-Seq
Genomic DNA was extracted from 2.5 x 107 cells using 950 µL of extraction buffer (20 mM Tris HCl (pH 8.0), 10 mM EDTA, 100 mM NaCl and 0.5% SDS) followed by RNaseA/T1 (Thermo Scientific) treatment and phenol/chloroform extraction. gDNA were sonicated and diluted to 17.5 mg/mL in CAP100 buffer (12.5% glycerol, 0.1% Triton-x-100, 20mM HEPES pH 7.9 and 100mM NaCl). NeutrAvidin Agarose Resin (Thermo Scientific) was conjugated with biotinylated hKDM2B ZF-CxxC protein (kindly provided by Rob Klose). 500 mL of diluted sonicated DNA were incubated with conjugated CxxC resin for 1 hour at 4°C. Serial washes and elutions using increasing salt concentration were performed. First, two washes with 1 mL of CAP100 buffer were carried out, followed by two elutions with 50 µL of CAP300 (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9 and 300 mM NaCl) and two elutions with 50 µL of CAP500 (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9 and 500 mM NaCl). Subsequently, the elution process was repeated using buffers with 700 mM and 1 M NaCl sequentially. DNA purification from eluted samples were performed using QIAquick PCR purification kit (Qiagen). Samples derived from elutions using 700 mM and 1 M NaCl were used for sequencing. NEBNext Ultra II DNA Library Prep kit for Illumina (New England Biolabs).