ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs
ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs
Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. The protocol was adapted from Groh et al., 2021 (PMID: 34650047) and Navarro et al., 2020 (PMID: 33037201). Cells were harvested and crosslinked in 3 ml of pre-tempered (25°C) ES medium supplemented with 1% formaldehyde (106 cells/3 ml) for 10 minutes at RT, with rotation. Formaldehyde was then quenched with glycine (125 mM final) and incubated 5 minutes at RT, with rotation. Cells were washed twice with ice-cold PBS containing 10%FBS (centrifugation at 1100 rpm for 4 minutes, at 4°C). Pellets can be stored at -80°C for several months. To prepare lysates, fixed cells were resuspended in 300 µl of Sonication Buffer 1 (50 mM Tris-HCl pH 8.0; 0.5% SDS) and incubated for 10 minutes on ice. Sonication was performed with Bioruptor Pico (Diagenode) for 20 cycles (30''ON/30''OFF). Sonicated lysates were then diluted 1:6 with lysis buffer (10 mM Tris-HCl ph 8.0; 100 mM NaCl; 1% Triton-X; 1 mM EDTA; 0.5 mM EGTA; 0.1% NaDoc; 0.5% N-laurolsarcosine) and the soluble fraction was collected after centrifugation at full speed for 10 minutes at 4°C. A 2.5% fraction of the supernatant was set aside for input and the fragmentation pattern (~150-500 bp) was checked by agarose gel electrophoresis. Lysates can be aliquoted and stored at -80°C with 10% glycerol (final). For each IP, 30 µl Protein G Dynabeads (Invitrogen) were washed twice with 1 ml PBS-T (PBS + 0.01% Tween-20) and incubated with the desired antibody for at least 1 hour at RT (or > 1 hour at 4°C). Coated beads were then washed (1X with PBS-T; 2X with lysis buffer), resuspended in 30 µl of lysis buffer/IP and added to the desired amount of chromatin (generally 15-25 µg of chromatin were used, corresponding to 2-4x106 cells). For ChIP-Rx, spike-in chromatin was prepared from Drosophila Schneider 2 (S2) cells following the same protocol and aliquots were stored at -80°C with 10% glycerol. Before immunoprecipitation, 3% of exogenous chromatin was added to each reaction. After overnight incubation at 4°C with rotation, beads-immunocomplexes were washed twice-each time for 5 minutes-with the following buffers: RIPA; RIPA + 360 mM NaCl; LiCl buffer (10 mM Tris-HCl ph 8.0; 250 mM LiCl, 0.5% NP-40, 0.5% NaDoc; 1 mM EDTA) and finally quickly 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/protein digestion was performed on beads by adding 2 µl  RNaseA (10 mg/ml stock) incubated 30 minutes at 37°C, followed by addition of 1.5 µl Proteinase K (20 mg/ml stock) incubated 1h @ 55°C. Reverse cross-link was performed with overnight incubation at 65°C. DNA was purified with 1.4X SPRI-select beads or QIAquick PCR Purification kit (Qiagen). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) and, after Qubit quantification, the quality of individual libraries was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.