title: GSM4190295: H3K9me3_clr4Δ_rep2; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM4190295