GSM6210099: PRMT1fl/fl; cx3r1cre-ert2 microglia normal diet_rep1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation for histone modification H3K27ac and was performed as follows, using ~500,000 microglia per assay and n=2 independent biological replicates per conditions. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4 °C, and pellets were resuspended in 200ul of sonication/ immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 sec “sonication-off”.  Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 RCF at 4 °C. Two microliters of supernatant were then set aside for input library sequencing controls. Supernatants were then immunoprecipitated on a rotator for two hours at 4 °C with H3K27ac antibody (Active Motif, 39685, 2.5 ug per sample) pre-bound to 17 ul Protein A Dynabeads (Life Technologies 10001D).   Immunoprecipitates were washed three times each on ice with ice-cold wash buffer I (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA pH 8.0 in water), wash buffer III (10 mM Tris-HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1mM EDTA in water) and TET (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.1% Tween-20, in water) and eluted with 1% SDS/TE at RT in a final volume of 100 ul. Reverse-crosslinking was then performed on immunoprecipitates and saved input aliquots. First, 6.38 ul of 5 M NaCl (final concentration 300 mM) was added to each sample immersed in 100 ul SDS/TE. Crosslinking was then reversed by overnight incubation at 65 °C in a hot air oven. Potentially contaminated RNA was then digested for one hour at 37 °C with 0.33 mg/ml RNase A, proteins were digested for one hour at 55 °C with 0.5 mg/ml proteinase K, and DNA was extracted using Sera-Mag SpeedBeads (Thermo Scientific, 6515205050250). Sequencing libraries were prepared from recovered DNA (ChIP) or generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex, Bioo Scientific) (Gosselin et al., 2014; Gosselin et al., 2017). Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-400 bp for ChIP-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX).  ChIP-seq librairies were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer's instruction.