GSM3759268: Chtop Input; Homo sapiens; ChIP-Seq
Cell pellets were lysed in ChIP Lysis Buffer 1 (50 mM HEPES-NaOH pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, protease inhibitors SigmaFAST) and rotated at 4°C for 5 minutes. Nuclei were then pelleted via centrifugation (3000 x g, 5 minutes at 4°C), and resuspended in ChIP Buffer 2 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, protease inhibitors SigmaFAST), before rotation at room temperature for 10 mins. Nuclei were pelleted via centrifugation (1500 x g, 5 minutes at 4°C) and resuspended in ChIP Lysis Buffer 3 (10 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors SigmaFAST). Samples were sonicated using a Bioruptor (High, 20 x [30s-ON/30s-OFF]) to generate chromatin fragments of 250-300 nts, and cleared by centrifugation (16100 x g, 15 minutes, 4°C). Lysate concentrations were measured by Bradford assay, and equal concentrations of chromatin were incorporated into the IPs. IPs were carried out overnight at 4°C using 5 µg of FLAG antibody (Sigma). 100 µL blocked protein-G Dynabeads were then added to the samples and incubated for 2 hours at 4°C. Following incubation, beads were washed with 4 x 0.5 mL RIPA Wash Buffer (50 mM HEPES-NaOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP40, 0.7% Na-deoxycholate, 0.1% N-lauroylsarcosine) and once with 0.5 mL ChIP Final Wash Buffer (10 mM Tris-HCl pH7.3, 1 mM EDTA, 50 mM NaCl). Complexes were eluted by adding 200 µL of ChIP Elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) and incubated at 65°C for 30 minutes. NaCl was added to a final concentration of 200 mM and cross-links were reversed overnight at 65°C. Samples were then treated with RNase A (0.2 mg/mL) for 2 hours at 37°C, followed by proteinase K (0.2 mg/mL) for 2 hours at 55°C. DNA was purified via phenol-chloroform extraction and ethanol precipitation, and then resuspended in water. Library construction was using the standard ChIP-seq protocol of Novogene (Beijing, China)
GEO Accession
GSM3759268