title: MP636_H3k9me2_rep1
organism: Schizosaccharomyces pombe
description: Protocols: S. pombe cells were cultivated to exponential growth phase in complex YES medium. At OD 0.5, 400 ml yeast culture was fixed by adding formaldehyde to final concentration of 1%. After 30 min incubation, remaining formaldehyde was quenched by 125 mM glycine for 10 min. Cells were washed with PBS and broken with glass beads in Lysis Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, FY protease inhibitors [Serva], 1 mM PMSF) using the FastPrep24 machine (MP Biomedicals). Extracted chromatin was sheared with the Bioruptor sonicator (Diagenode) using 15 (biol. rep. 1-3) or 30 cycles (biol. rep. 4) of 30s on, 30s off on high power settings. For each immunoprecipitation (IP) within a biological replicate we used the same amount of chromatin extract (in mg of total protein, range 2.5 - 3.7 mg between replicates), for input DNA control 1/10 of chromatin extract amount was kept. For each IP 5 ug antibody (H3: Ab1791, H3K9ac: Ab4441, H3K9me2: Ab1220, all Abcam) were incubated with chromatin extract for 1 hour at 4°C. Then, 50 ul BSA-blocked Protein A-coated magnetic beads (ThermoFisherScientific, 10002D) were added to chromatin extract-antibody suspension and incubated for further 4 hours at 4°C. Crosslinks were reversed in both the precipitated material and input chromatin extract, and samples were treated with RNase A and proteinase K. DNA was purified using phenol-chloroform extraction and sodium acetate/ethanol precipitation. Concentration of DNA was measured by Quantus and size distribution was checked on Agilent Bioanalyzer using the High Sensitivity DNA Assay. In biological replicate #4, purification on Agencourt AMPure XP beads (Beckman Coulter, AC63880) was performed following the phenol-chloroform extraction to remove low-molecular fragments/RNA. Library construction was performed by BGI Tech Solutions (Hong Kong), according to the company's standard BGISEQ-500 ChIP-seq procedure.