ChIP-seq in Bl6xSpretus F1 hybrid mESCs
ChIP-seq in Bl6xSpretus F1 hybrid mESCs
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.  The protocol was adapted from (Trovato et al., 2024), with minor modifications. 10 million mESCs (i.e. DNMT TKO cells for H3K27Ac and Bl6xSpret F1 hybrid cells for TFs) were harvested and cross-linked in 3 ml pre-tempered (25°C) ES medium containing 1% formaldehyde and supplemented with sodium butyrate (5mM final concentration) for 10 min at room temperature with rotation. The cross-linking reaction was quenched by adding glycine (125mM final concentration) and incubated for 10 min at room temperature. Cells were washed twice with ice-cold PBS containing 10% FBS and centrifuged at 200g for 5 min at 4°C. At this point, cell pellets can be snap frozen and stored at –80 °C for several months. Pellets were resuspended in 300 µl Sonication Buffer (50 mM Tris-HCl pH 8.0; 0.5% SDS) and chromatin was sheared using the Bioruptor Pico (Diagenode) for 15 cycles (30” ON/30” OFF). Sonicated lysates were diluted 1:6 in 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 min 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 3 µg antibody against H3K27ac (ab4729, Abcam), 5 µg antibody against KLF4 (AF3158, R&D Systems), CTCF (07-729, Merck Millipore) or Sox2 (AF2018, R&D Systems) for 1 h at room temperature with 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 chromatin (15 µg for H3K27Ac; 10 µg for TFs). At this point, for the H3K27Ac ChIP, 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 min 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 for 30 min at 37 °C, followed by the addition of 1.5 µl of Proteinase K (20 mg/ml stock) and incubation at 55 °C for 1 h. 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)