GSM5321296: Input_wildtype_1; Schizosaccharomyces pombe; ChIP-Seq
100 mL yeast cultures were grown to mid-log phase to OD600 = 0.6. The cultures were cooled down at RT for 10 min and fixed with 1% FA for 20 min at RT on the shaker. Cross-linking was stopped with 150 mM Glycine for 10 min at RT. Cells were washed 2x with 30 mL ice-cold dH2O, the pellet was frozen in liquid nitrogen and kept at -80°C until further processing. Pellets were resuspended in 1 mL FA(1) buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (v/v), 0,1% NaDeoxycholate (w/v), 0,1% SDS (w/v), supplemented with Roche protease inhibitors. Cells were broken in a bead beater (Precellys): 9x30s. After centrifugation, the supernatant and the pellet were sonicated for 15 min at 4°C. Chromatin extracts were spun down for 10 min at 14000 rpm at 4°C. 500 µl chromatin was used and 1 µl of H3K9me2 antibodies (Abcam, Ab1220). Samples were incubated with antibodies O/N at 4°C. 25 µl of FA(1)-washed Dynabeads were added to each sample and they were incubated for 2 hours at 4°C. Samples were then washed 3x for 5 min at RT with FA(1) buffer, FA(2) buffer (FA(1) buffer with 500 mM NaCl), once with LiCl buffer (10 mM TrisHCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0,5% NP-40 (v/v), 0,5% NaDeoxycholate (w/v)) and once with TE buffer. DNA was eluted from the antibodies with ChIP Elution buffer (50 mM Tris HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 15 min at 65°C. DNA was treated with RNAseA, Proteinase K and de-crosslinked O/N at 65°C. DNA was purified with Zymo Research ChIP DNA Clean and Concentrator kit according to the manual instructions. To obtain enough material for the library preparation three technical IP replicates were pulled. ChIP-seq libraries were prepared with 2 ng of DNA with NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manual instructions. The libraries were sequenced at LAFUGA at the Gene Center (LMU).