GSM5710316: H1 Day 4 (Time Point)- KDM1A ChIP (Experiment#1); Homo sapiens; ChIP-Seq
Briefly, about 8×106 hESCs and 5×106 hESC-derived cardiomyocytes were used for each immunoprecipitation. Cells were detached using Accutase, fixed in PBS containing 1% formaldehyde and quenched with 125 mM glycine. Cells were lysed in ChIP lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl at pH 8.1) and sonicated (E220evolution Focused-ultrasonicator, Covaris) to generate 250-bp DNA fragments. Soluble chromatin was diluted eightfold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl at pH 8.1, and 167 mM NaCl). Chromatin was incubated overnight at 4°C with KDM1A or T7-tag antibodies and recovered using Dynabeads Protein G (Thermo Fisher Scientific). Beads were washed sequentially with buffers at different salt concentrations: low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl), lithium chloride wash buffer (0.25 M lithium chloride, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and TE buffer. Immunocomplexes were eluted in ChIP elution buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl), at 30°C for 20 minutes and cross-linked overnight at 65°C. DNA was purified using QIAquick PCR columns (Qiagen) according to the manufacturer's instructions. DNA libraries were generated using the MicroPlex Library Preparation Kit v2 (Diagenode) according to the manufacturer's instructions ChIP-seq sequencing has been performed on Illunima Hiseq 4000 or Novaseq 6000 SP1 with 150 bp paired-edn method