H3K27ac ChIP-seq of human HepG2 cells
Formaldehyde was added directly to the culture media of flasks containing 10x106 HepG2 cells, to a final concentration of 1%. After 10 min incubation at room temperature, fixation was quenched by adding 125 mM ice-cold glycine, and flasks were washed three times with ice-cold phosphate buffered saline (PBS). Cells were then scraped and washed three more times with PBS, each washing step being followed by sedimentation at 1,300 x g. Pellets were resuspended in 1mL lysis buffer (5 mM PIPES-pH=8.0, 85 mM KCl, 0,5% NP-40, cOmplete Mini cocktail tablets (Roche) and incubated at 4°C for 15 mins with vortexing. Lysed cells were sedimented at 13,000 x g, resuspended in 500 µL of sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl-pH=8.0, cOmplete Mini cocktail tablets) and sonicated on ice with an MSE sonicator (6 pulses of 15s each at 15% amplitude with 30s off between each pulse). The generated fragments were approximately 500 bp long, as determined experimentally. After sedimentation at 13,000 x g, the supernatant was transferred into a new tube, diluted ten times with IP buffer (0,01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH = 8.0), 167 mM NaCl) and supplemented with cOmplete Mini cocktail tablets. For each 500 µL extract, a mixture of 60 µL of Dynabeads protein A and 60 µL of Dynabeads protein G (ThermoFisher), conjugated with 2 µg anti-H3K27ac rabbit polyclonal antibody (Abcam ab4729) was added. After incubation at 4°C overnight, the beads were subsequently washed with buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH = 8.0), 0.15 M NaCl), buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH = 8.0), 0.5 M NaCl) and buffer C (0,25 M LiCl, 1% NP40, 1% Na-deoxycholate, 1 mM EDTA, Tris-HCl (pH = 8.0), at 4°C with permanent rotation for 5 mins. Following two washes with TE buffer (10 mM Tris-HCl, 10 mM EDTA, pH = 8.0), samples were eluted in 200 µL freshly prepared NaHCO3 (0,1 mM) supplemented with 1% SDS. Cross-links were reversed by overnight incubation at 65°C. Samples were incubated with 20µg/mL RNAse at 37°C for 1 hour, then proteinase K was added to 20 g/mL and the mixture was incubated for 2 hours at 45°C. DNA was purified using High pure PCR template preparation kit (Roche). Libraries were prepared from 5 ng DNA with Ovation Ultralow Library Systems (Nugen) according to manufacturer’s instructions. Libraries were sequenced on Illumina HiScanSQ.