title: GSM3383801: hop1 red1 ChIP 2h; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: ChIP was performed as described (Aparicio et al., 2004; Mendoza et al., 2009; Metivier et al., 2003; Panizza et al., 2011) with modifications. Rec114-Myc diploids were sporulated and 50 mL of meiotic culture (2 x 10^9 cells) was collected at different times 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 μg/mL aprotinin, 1% protease inhibitor cocktail (Sigma), 1x Complete Protease Inhibitor Cocktail (Roche)) with ca. 900 μL of 0.5 mm zirconia/silica beads (BioSpec Products) in 2 mL screw-cap tubes. Cells were disrupted by vigorous shaking at 6.5 m/s for 1 min x 10 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 3 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 3–5 times to yield average DNA size less than 500 bp. WCE was flash frozen and stored at -80°C.
identifiers.namespace: GEO
identifiers.id: GSM3383801