title: GSM1432450: wt_input; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: 700 OD600 of cells were fixed in 1% w/v formaldehyde for 25 min at room temperature. The reaction was stopped by adding glycine to a final concentration of 125 mM. After extensively washing the pellets in Tris-buffered saline solution, the cells were aliquoted into 14 equal samples. Cells were lysed by bead beating in 0.5 mL per sample of 50 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1% v/v Triton-X 100, 1 mM PMSF, 1× Complete Protease Inhibitor Cocktail. Following addition of Triton X-100 to 1% v/v, chromatin was sheared by sonication in a Misonix Sonicator 3000 (Farmingdale, NY, 15 cycles of 30 s ON at maximum intensity and 1 min OFF). The lysate was cleared by centrifugation and adjusted to 10 mL at 15 mg mL-1 of total protein, as determined by the Bradford assay. This lysate was incubated with 0.5 mL of pre-equilibrated IgG resin (GE Healthcare Life Sciences) overnight at 4 °C with rotation. Next, the resin was washed three times with 1 mL of the lysis solution described above without protease inhibitors, once with 50% the same solution and 50% of 20 mM HEPES pH 7.6, 150 mM NaCl, 0.1% v/v NP-40, 1 mM EDTA and once with 100% of the latter solution. The IgG resin was resuspended with 0.75 mL of the latter solution supplemented with 0.75 mM DTT and 500 U of TEV protease (Eton Biosciences, San Diego, CA) and incubated at room temperature for 4 h with rotation. The resin was washed three times with 0.5 mL of the same solution used for proteolysis, which were combined with the eluate, supplemented with 0.2 mL of pre-equilibrated anti-FLAG magnetic resin (M2, Sigma-Aldrich), and incubated overnight at 4 °C with rotation. The anti-FLAG resin was washed three times for 10 min with 10 mL of 50 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA, 1 % v/v Triton-X 100, 0.1% v/v sodium deoxycholate, twice more in the same buffer but containing 500 mM NaCL, twice in 10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% v/v NP-40, 0.5% v/v sodium deoxycholate and once in Tris-EDTA solution. The resin was eluted twice with 0.5 mL of 10 mM Tris pH 8, 1 mM EDTA, 1% v/v SDS for 15 min at 70 °C. The eluted DNA was de-crosslinked overnight at 65 °C, purified with Qiagen’s PCR Purification Kit (Qiagen, Germantown, MD), and eluted in 50 µL of TE. Libraries were prepared from 10 ng of DNA using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommended protocol. The libraries were amplified with 18 cycles of PCR and size selected on a 2% agarose gel to remove adapter dimer products. The purified DNA library was denatured in 0.1 N NaOH and diluted to a final concentration of 10 pM before being loaded onto an Illumina single read flow-cell.