title: GSM8602867: H3K9me3_raf1R576H_upf1∆_FE_Exp4_Rep2; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: For the H3K9me3 chromatin immunoprecipitation (ChIP) assay, 5x108 cells were crosslinked with 3% paraformaldehyde (PFA) for 30 minutes at room temperature, followed by quenching with 125 mM glycine. For Swi6 or Raf2 ChIPs,1x109 cells were incubated for 2 hours at 18°C, fixed in 3% PFA, and washed in ice-cold PBS. Cells were treated with 10mM Dimethyl adipimidate (DMA) at room temperature for 45 minutes. Fixed cells were resuspended in ChIP lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) supplemented with 1 mM PMSF and protease inhibitors (Roche cOmplete, Mini Protease Inhibitor Cocktail) and lysed by bead-beating ( Biospec Mini-Beadbeater-16). The lysates were sonicated with a Diagenode Bioruptor for 14-cycles on medium power setting (30 s ON, 30 s OFF) to shear DNA into 0.4–0.6 kb fragments. Cellular debris were removed by centrifugation and 2% supernatants was kept aside for whole cell extract input control. The remaining lysates were pre-cleared with agarose beads for 1 hour at 4°C. After bead removal, lysates were incubated with the appropriate antibody (2–10 μg) overnight at 4°C with slow rotation. Antibodies used includes anti-H3K9me3 (Abcam), anti-c-Myc (Santa Cruz), and home-made affinity-purified anti-Swi6. Pre-washed protein A and protein G agarose beads were added to the lysates and incubated for 4 hours at 4°C. The beads were sequentially washed, twice with Wash Buffer I (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), twice with Wash Buffer II (50 mM HEPES/KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), twice with Wash Buffer III (10 mM Tris/HCl pH 8.0, 250 mM LiCl, 0.5% IGEPAL, 0.5% sodium deoxycholate, 1 mM EDTA), and once with TE buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA). The chromatin was eluted from the beads twice with 50 μl of elution buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65°C for 30 minutes each, with shaking (~1,200 rpm). Eluted chromatin and input control samples were reverse crosslinked overnight at 65°C. Samples were treated sequentially with RNase A (10 μg, 2 hours, 37°C) and Proteinase K (20 μg, 2 hours, 37°C), and subsequently purified using the Qiagen PCR purification kit. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer's protocol and analyzed using an Agilent 4200 TapeStation system (Agilent).