P-GSE67912-2 -

nucleic acid library construction protocol
ChIP was performed as described (Murakami et al., 2014). Cells were sporulated and 50 mL of meiotic culture (2 x 109 cells) was collected at 4h and crosslinked with 1% formaldehyde for 30 min at room temperature. Crosslinking was terminated by 5 min incubation with 131 mM glycine. Cells were washed twice with ice-cold TBS, flash frozen with liquid nitrogen and stored at -80°C. Cells were suspended in 1 mL of Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, 7 ug/mL aprotinin, 1% protease inhibitor cocktail (Sigma), 1x Complete Protease Inhibitor Cocktail (Roche)) with ca. 900 uL of 0.5 mm zirconia/silica beads (BioSpec Products) in 2 mL screw-cap tubes. Cells were disrupted by vigorous shaking 3 min x 6 times in Mini-beadbeater-16 (BioSpec Products) or at 6.5 m/s for 1 min x 8 times in FastPrep24 (MP Biomedicals). 1 mL of Lysis buffer was added after cell disruption. Subsequently, chromatin in the whole cell extracts (WCE) was sheared by sonication with "M" intensity, 30 sec ON/ 30 sec OFF for 15 min x 2 times in Bioruptor Sonication System UCD200 (Diagenode) in 15 mL polystyrene conical tubes. Insoluble fraction (cell debris) was removed by centrifugation at 21,130 g, 5 min, 4°C. WCE was further sonicated with the same conditions 1-3 times to yield average DNA size less than 500 bp. WCE was flash frozen and stored at -80°C. The ChIPseq library was prepared following Illumina standard protocol. We used Trueseq DNA sample prep kit.
Experiment E-GEOD-67912