chip_k27_r2_tko_dmso
Mus musculus
Protocols: 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).
ENA first public
2026-02-02
INSDC center name
European Molecular Biology Laboratory (EMBL)
INSDC status
public
Submitter Id
E-MTAB-14916:chip_k27_r2_tko_dmso
broker name
ArrayExpress
cell_line
159 DNMT TKO
cell_type
mouse embryonic stem cell
collection date
not collected
common name
house mouse
developmental stage
mouse embryo stage
disease
normal
genetic modification
gene knock out
genotype
CRISPR-KO of the three DNA methyl transferases (DNMT TKO)
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
embryonic tissue
sample name
E-MTAB-14916:chip_k27_r2_tko_dmso
scientific_name
Mus musculus