H3K27ac ChIP-sequencing of fixed or live FACS-sorted GLF16+ and GLF16- cells
Li-Fraumeni-p21WAF1/Cip1 Tet-ON cells were seeded onto 10-cm cell culture plates and once they reached 70% confluency they were treated with 10 microgram/ml Doxycycline for 6-8 days so that cells become senescent. Untreated cells were also cultured to serve as negative and unstained controls during the sorting analysis.Cells were either fixed (1% formaldehyde, 30 min; 0.125M glycine, 5 min) and stained with GLF16, or inoculated with m-GLF16 for 3h as described above. In both cases, GLF16+ cells were sorted against GLF16- using FACSAria II and collected in 5ml sterile, RNAse-free tubes. After cell and nuclei lysis, chromatin was sonicated, incubated overnight with anti-H3K27ac antibody (Abcam, ab4729). Following immobilization on protein G beads and washing steps, samples were treated with proteinase K, crosslinks were reversed overnight at 65C and the DNA was purified. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Samples were sequenced with a NovaSeq 6000 Illumina sequencer at the Greek Genome Center situated at BRFAA.