GSM7661012: SV20 ME, H3K27ac, ChIP; Homo sapiens; ChIP-Seq
1 million (histone modification ChIP-seq) to 10 million (transcription factor ChIP-seq) cells were cross-linked with 1 % fresh methanol-free formaldehyde for 10 min at room temperature (RT). The cross-linking was stopped with the addition of fresh glycine to a final concentration of 0.125M and incubation for 5 min at RT. Cells were subsequently washed twice with cold PBS, pelleted and resuspended in cold ChIP lysis buffer and lysed for 15 min on ice while dounce homogenizing 20x during the incubation. After re-pelleting, nuclei were resuspended in ChIP nuclear lysis buffer for 30-60 min on ice. Chromatin was sheared by sonication for 20 cycles at 60 hz for 30 sec on/30 sec off. Cellular debris was removed by centrifugation. Chromatin was pre-cleared by rotating at 4 °C for 1h with Protein A/G Ultralink beads (Thermo Fisher Scientific, Pierce) in ChIP dilution buffer. Chromatin was incubated with primary antibodies overnight at 4°C while rotating. Protein A/G Ultralink beads (Thermo Fisher Scientific, Pierce) were added to the immunoprecipitation (chromatin + antibody) mix and incubated at 4°C for further 4 hours. Unbound fraction of the IgG chromatin solution was kept as input DNA. Beads were subsequently washed for 5 min at 4°C once each in low salt buffer, high salt buffer, lithium chloride buffer, followed by two washes in TE buffer, before eluting the DNA by incubating the beads in 125µl-250µl fresh elution buffer for 15 min at RT while vortexing/rotating. To reverse the cross-linking, samples were incubated overnight at 65°C in 0.192M NaCl, 40mM Tris-Cl, pH6.5, 10mM EDTA, 0.08mg/ml Proteinase K and 0.1 mg/ml RNaseA. DNA was subsequently extracted and purified by phenol-chloroform extraction and precipitated in Ethanol at -80°C before being resuspended in 50µl of H2O. samples were library prepped using the NEBNext Ultra II DNA library Prep Kit for Illumina following manufacturer's recommendations