GSM4190292: H3K9me3_grc3V70M_rep2; Schizosaccharomyces pombe; ChIP-Seq
50 OD600 units of cells per ChIP were fixed with 1% formaldehyde for 15min, quenched with glycine and collected. Cell pellets were resuspended in lysis buffer (50mM  Hepes/KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.1% Na Deoxycholate, 0.1% SDS, 1mM PMSF, supplemented with protease inhibitors) and cells were lysed by the glass bead method, using MagNA Lyser (Roche) (3 pulses of 90s at 4500rpm, 1 pulse of 45s at 5000rpm). The lysates were sonicated by 3 pulses of 20s (amplitude 40%) in a Branson microtip sonifier with intermittent cooling on ice, to a fragment distribution of 200-1000 bp. Cell debris were removed by centrifugation (16000xg) at 4*C for 15min and the cleared lysates were incubated for 2 hours at 4*C with magnetic beads coupled to antibodies against the targets of interest. For H3K9me2 30µl Protein A Dynabeads (Invitrogen) were coupled with 2µg anti-H3K9me2 antibody (Ab1220, Abcam). For H3K9me3 ChIPs, 30µl Streptavidin Dynabeads M280 (Invitrogen) were coupled to 2µg of anti-H3K9me3 antibody (Diagenode C15500003) and blocked with 5µM biotin. For Crb3-TAP ChIPs, 4.5µg Dynabeads M-270 Epoxy beads (Invitrogen) were coupled to 1.5µg rabbit IgG (Sigma, #15006) and incubated with the cleared lysate. The beads-protein complexes were washed three times with lysis buffer, once with chilled TE buffer (10mM Tris/HCl, 1 mM EDTA) and eluted in elution buffer (50mM Tris/HCl pH 8.0, 1mM EDTA, 1% SDS) with heating (65oC) for 20min. Crosslinks were reversed at 65*C overnight and the eluates were treated with RNase A and Proteinase K (Roche). For ChIP-seq, reverse crosslinked DNA treated with RNase A and proteinase K was purified using the Qiagen PCR purification kit. The DNA was then sonicated in a QSonica water bath sonicator (5 min with 15s ON/OFF pulses, 20% amplitude) to a fragment size of ~200bp, concentrated and quantified using Qubit dsDNA high sensitivity kit. 1-10ng of DNA were used to prepare libraries as described previously (Wong et al, 2013). Libraries were pooled and sequenced on Illumina HiSeq 2500 and Illumina NextSeq 500 platforms.
GEO Accession
GSM4190292