GSM2174863
GSM2174863: ura4+::KAN leo1∆ + empty vector (spy6421 + pDM829); Schizosaccharomyces pombe; ChIP-Seq
Cell pellets were resuspended in 600 μl lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS, 1 mM PMSF, protease inhibitor tablet (Roche)), and disrupted by bead beating (MagNA Lyser, Roche) for 3x90 sec at 4500 rpm with 0.5 mm glass beads. Tubes were punctured and the flow-through was collected in a new tube by centrifugation. After sonication for 3x20 sec at 50% amplitude (Branson Digital Sonifier), the extract was centrifuged (Eppendorf 5415R) for 15 min at 13,000 rpm. The soluble chromatin was then transferred to a fresh tube. H3K9me2 (Abcam, Ab 1220) was preincubated with Dynabeads Protein A, and for each immunoprecipitation, 2 μg antibody bound to 30 μl beads was added to soluble chromatin. Samples were incubated for 2 hours at 4°C with rotation, after which the beads were collected on magnetic stands, and washed 3 times with 1 ml lysis buffer and once with 1 ml TE, and eluted with 50 μl preheated buffer (50 mM Tris.HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65˚C for 15 min. The eluate was collected, and 75 μl 1XTE/0.67% SDS was added on the beads, the incubation was repeated and both elutions were combined. Immunoprecipitated samples were incubated overnight at 65˚C in the presence of 0 μg RNase A to reverse crosslink and RNase treated the samples, respectively. 5 μl proteinase K (Roche) was added and incubation was continued at 55°C for 1 h. Samples were purified using PCR purification kit (Qiagen). Libraries for Illumina sequencing were constructed following the protocol detailed in Wong et al, 2013 (Wong, K. H., Jin, Y. and Moqtaderi, Z. 2013. Multiplex Illumina Sequencing Using DNA Barcoding. Current Protocols in Molecular Biology. 101:7.11:7.11.1–7.11.11.), starting with ~10 ng of immunoprecipitated DNA fragments. Each library was generated with custom-made adapters carrying unique 6 nt barcode sequences at the ligating end. Barcoded libraries were mixed and sequenced with Illumina HiSeq 2000.
GEO
GSM2174863
GEO Accession
GSM2174863