Rabbit anti h3k27me3 antibody

Sorted cells were centrifuged in a swing-out rotor at 4,000 g for 15 min at 4°C, transferred to a thin-walled 0.5 ml microfuge tube (Axygen PCR-05-C), re-centrifuged and then resuspended in 130 μl Lysis Buffer (17 mM Tris.HCl pH 8, 3.4 mM EDTA.Na₂, 0.34% SDS) containing protease inhibitors (Sigma-Aldrich P8340). The lysate was sonicated for 5 cycles at high setting using a Diagenode Bioruptor (1 cycle is 30 sec ON and 30 sec OFF). After sonication, the sample was centrifuged at 16,000 g for 15 min at 4°C, the chromatin-containing supernatant transferred to a fresh microfuge tube and 70 μl RIPA Buffer (36.7 mM Tris.HCl pH 8, 2.5 mM EDTA.Na₂, 0.01% SDS, 2.46% Triton X-100, 374 mM NaCl) containing protease inhibitors added to the chromatin sample. The ChIP reaction, washes and DNA purification were performed as in Dahl and Collas [56 (link),57 (link)]. In brief, magnetic beads were coated with 2.4 μg of rabbit anti-H3K27me3 antibody (Millipore 07–449) and incubated overnight in a volume of 100 μl with chromatin from ~100,000 YFP⁺ sorted cells. Beads were washed, chromatin eluted, RNA and proteins digested, the DNA purified by phenol/chloroform extraction followed by ethanol precipitation using linear acrylamide as carrier and resuspended in 10 μl PCR grade water. Approximately 5 μl of chromatin was retained as input and purified alongside the ChIP sample.