ChIPseq in 159 DNMT TKO mESCs upon p300 inhibition
ChIPseq in 159 DNMT TKO mESCs upon p300 inhibition
Mouse ES cells (129 WT, DNMT TKO, TET TKO and F1 hybrid cells (129/CAST)) were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% Fetal Bovine Serum (FBS), LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37°C and 5% CO2. Medium was changed daily and cells were split every second day. p300 inhibition was performed by adding 3 μM A-485 (Selleck Chemicals, S8740) to the ES medium for 24 hours. The protocol was adapted from [Trovato et al., 2024],with minor modifications. 10^6 mESCs were harvested and cross-linked in 3ml pre-tempered (25°C) ES medium containing 1% formaldehyde for 10 minutes at room temperature, with rotation, supplemented with sodium butyrate (5mM final concentration). The cross-linking reaction was quenched by adding glycine (125mM final concentration) and incubated for 10 minutes at room temperature. Cells were washed twice with ice-cold PBS containing 10% FBS and centrifuged at 200g x 5mintes at 4°C. Cell pellets can be snap frozen and stored at –80 °C for several months. Pellets were resuspended in 300 µl of Sonication Buffer (50 mM Tris-HCl pH 8.0; 0.5% SDS) and chromatin was sheared with Bioruptor Pico (Diagenode) for 15 cycles (30” ON/30” OFF). Sonicated lysates were diluted 1:6 with lysis buffer (10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1% Triton X-100; 1 mM EDTA; 0.5 mM EGTA; 0.1% sodium deoxycholate; and 0.5% N-lauroylsarcosine). After centrifugation at full speed for 10 minutes at 4 °C, the soluble fraction was collected. A 2.5% portion of the supernatant was kept as input, and the fragmentation pattern (~150–500 bp) was analyzed by agarose gel electrophoresis. At this point, sonicated lysates may be aliquoted and stored at –80 °C with a final concentration of 10% glycerol. For each immunoprecipitation (IP), 30 µl of Protein G Dynabeads (Invitrogen) were washed twice with 1 ml of PBS-T (PBS + 0.01% Tween-20) and incubated with the antibody against H3K27ac (ab4729, Abcam) at 1 hour at room temperature, on rotation. Coated beads were washed once in cold PBS-T and twice in lysis buffer, then resuspended in 30 µl of lysis buffer per IP and added to 15 µg of chromatin. At this point, 1 µg of exogenous chromatin (from Drosophila Schneider 2 (S2) cells, prepared with the same protocol and stored at –80 °C with a final concentration of 10% glycerol) was added to each reaction as spike-in.  After overnight incubation at 4 °C with rotation, bead-immunocomplexes were washed twice (5 minutes per wash), using the following buffers: RIPA, RIPA supplemented with 360 mM NaCl, and LiCl buffer (10 mM Tris-HCl, pH 8.0; 250 mM LiCl; 0.5% NP-40; 0.5% sodium deoxycholate; 1 mM EDTA). The complexes were then briefly rinsed with TE buffer and eluted in ChIP SDS elution buffer (10 mM Tris-HCl, pH 8.0; 300 mM NaCl; 5 mM EDTA; 0.5% SDS). RNA and protein digestion were performed by adding 2 µl of RNase A (10 mg/ml stock) and incubating at 37 °C for 30 minutes, followed by the addition of 1.5 µl of Proteinase K (20 mg/ml stock) and incubation at 55 °C for 1 hour. Cross-links were reversed by overnight incubation at 65 °C. DNA was then purified using 1.4X SPRI-select beads. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) and sequenced on Aviti Cloudbreak Low 2x150 (250 M clusters/run).