GSM5493771: SPJ649_WT_Input; Schizosaccharomyces pombe; ChIP-Seq
Log-phase yeast cells were crosslinked with 1% formaldehyde for 20 minutes with shaking at room temperature, followed by 5 minutes quenching with 125mM glycine. Cells were harvested, washed with PBS (phosphate-buffered saline) and flashfrozen with liquid nitrogen. The thawed pellet was washed and then resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholate, 1mM PMSF). Ice-cold glass beads were added and the mixtures were vigorously disrupted in a bead-beater with four 30-seconds rounds. The lysates were collected and NP buffer was added (10 mM Tris, pH 7.4, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2). MNase was added to the reaction and the reactions were incubated at 37°C for 20 minutes. MNase amount was titrated empirically so that the chromatin was digested to yield mainly mono- and di-nucleosomes. The reaction was stopped by the addition of 0.5 M EDTA, and the tubes were placed on ice. 5X ChIP lysis buffer was added to the reaction, mixed by short vertexing, and the tubes were incubated on ice for 30 minutes. The reactions were then cleared by centrifugation at 16,000 x g for 10 minutes. A small fraction of the cleared supernatant was reserved as input and the rest was used for immunoprecipitation. The protocols for immunoprecipitation, reverse-crosslinking, and DNA precipitation are the same as in the previous ChIP section. The precipitated DNA was treated with RNAase A (EN0531, Thermo Fisher Scientific) for 1 hour at 37°C. DNA concentration was determined with the Qubit dsDNA HS Assay Kit (Q33230, Thermo Fisher Scientific). 1-5 ng of ChIP and input DNA were used for library construction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB). Libraries were pooled and sequenced with single-end sequencing on a NextSeq500/550 at the JP Sulzberger Genome Center at Columbia University. 1-5 ng of ChIP and input DNA were used for library construction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB).