ChIP analysis H3K27 in the R 6/2 model
ChIP tests were performed on cross-linked chromatin samples, with the following modifications. The tissues were washed with a cold 1  PBS buffer, incubated for 10 minutes with 1% formaldehyde (FA) at room temperature, and washed again with 1  PBS. For ChIP against chromatin-modifying enzymes, double crosslinking with EGS (ethylene glycol bis (succinic acid N-hydroxysuccinimide ester; E3257; Sigma-Aldrich) was used: FA crosslinked tissue was incubated with EGS for 1 hour at room temperature with gentle agitation, were washed three times with cold PBS, resuspended in 1 ml of cell lysis buffer (5 mM HEPES, pH 8.0, 85 mM KCl, Triton X-100 and proteinase inhibitors) and homogenized with a Dounce homogenizer (Kimble, Vineland, NJ) (~ 25 times using a mortar.) The cell extract was collected by centrifugation at 3,000 g, resuspended in 0.3 ml of sonication buffer (50 mM HEPES, pH 7.9, 140 mM NaCl, EDTA 1 m, 1 % Triton X-100, 0.1 % deoxycholate acid, 0.1 % SDS and a mixture of proteinase inhibitors), and incubate for 10 minutes on ice Extracts from Chromatin sonicated for 15 minutes, 30 seconds, 30 seconds in BioruptorPico (Diagenode) and centrifuged at 16,000 g for 15 min at 4 C to obtain fragments of 500 bp or smaller. The supernatant was collected, divided into aliquots, frozen in liquid nitrogen, and stored at -80 C; an aliquot was used for quantification by Qubit fluorometric quantification (Thermo Fischer). Chromatin size was confirmed by electrophoretic analysis. Extracts of cross-linked chromatin (500 ng) were resuspended in sonication buffer to a final volume of 500 l; Samples were pre-cleaned by incubating with 24 g of normal immunoglobulin G (IgG) and 50 l of A / G-agarose protein microspheres (Santa Cruz Biotechnology) for 1 hour at 4 C with agitation for preclearance. The chromatin was centrifuged at 4,000 g for 5 minutes, and the supernatant was collected and immunoprecipitated with specific antibodies for 12-16 h at 4  C. The immune complexes recovered with an additional 50 l of magnetic beads (Dynabeads, Invitrogen), followed by incubation for 1 hour at 4 C with gentle agitation. The immunoprecipitated complexes were washed once with sonication buffer, with LiCl buffer (100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 0.1 % Nonidet P40 and 0.1 % deoxycholic acid), and once with Tris-EDTA buffer (TE) pH 8.0 (2 mM EDTA and 50 mM Tris-HCl, pH 8.0), each time for 5 minutes at 4 C; This was followed by centrifugation at 4,000 g for 5 min. Protein-DNA complexes were eluted by incubation with 100 l elution buffer (50 mM NaHCO3 and 1 % SDS) for 15 minutes at 65 C. Extracts were incubated for 12-16 h at 65 C, to reverse crosslinking the proteins digested with 100 g / ml proteinase K for 2 hours at 50 C, and the DNA was recovered by phenol/chloroform extraction and ethanol precipitation using glycogen (20 g / ml) as the precipitation vehicle.