GSM9361297: PolII_ChIP_5%_HD; Schizosaccharomyces pombe; ChIP-Seq
Fission yeast cells (approximately 5 x 108 cells at OD595 = 0.5) were fixed with 3% pFA at 26°C for 30 minutes, washed twice with 1´ PBS, and further fixed with 10 mM dimethyl adipimidate (Sigma Aldrich) at room temperature for 45 minutes.  After two washes with 1´ PBS, the cells were disrupted with glass beads using Mini-Beadbeater-16 (BioSpec Products) 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, 0.1 mM PMSF, and EDTA-free Protease Inhibitor Cocktail (Roche)].  Chromatin DNA was sheared into 100–500 bp fragments by Bioruptor UCD-200 (Diagenode), and the soluble fraction was collected by centrifugation at 13,000 rpm for 15 minutes.  A total of 50 μl ChIP lysis buffer containing 5 µl antibody and 15 µl Dynabeads protein G (Thermo Fisher Scientific) was added to the soluble fraction, and the bead suspension was incubated at 4°C for 2 hours with a gentle rotation.  The beads were washed twice with ChIP lysis buffer, once with ChIP lysis buffer containing 0.65 M NaCl, once with washing buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA), and once with TE (10 mM Tris-HCl pH 8.0, and 1 mM EDTA) for 5 minutes each with a gentle rotation.  The protein-DNA mixture was eluted by incubating the beads with TES (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 1% SDS) at 65°C for 30 minutes with harsh agitation at 1,000 rpm using ThermoMixer (Eppendorf) and subjected to reverse crosslinking at 68°C overnight in the presence of 0.5 M NaCl.  DNA was purified using QIAquick PCR purification kit (Qiagen) The recovered DNA was subjected to library construction using NEBNext Ultra DNA library prep kit (NEB).  The sequencing libraries were amplified by PCR using the NEBNext Ultra II Q5 Master Mix (NEB) with NEBNext Multiplex Oligos for Illumina (NEB).  The libraries were sequenced by NextSeq 2000 (Illumina) to obtain paired-end reads.