title: GSM1262377: G1_phase_synchronized_GCC biological replicate #2; Schizosaccharomyces pombe; OTHER
library_construction_protocol: Chromatin isolation and GCC were performed as in Rodley et al. 2009 and Cagliero et al. 2013, with modifications; Following synchronization, cultures (200 ml) were cross-linked, washed, and suspended in FA-lysis buffer. Aliquots containing ~9.5*108 cells were made up to a volume of 330 μl with FA-lysis buffer and the cell walls were digested with T20 Zymolyase (70 μl at 75mg/ml; 35°C, 40 min with periodic inversion) before heat inactivation (60°C, 5 min). Acid washed glass beads (500 μl) were added to each sample before disruption in a Geno/Grinder (-20°C; 1,750 rpm, 2 x 30 sec on 60 sec off; SPEX sample prep 2010). Glass beads were removed by the centrifugation of chromatin through a pin hole into a clean tube (2,000 rpm, 1 min). Chromatin was pelleted (13,000 rpm, 15 min, 4°C), washed with FA-lysis buffer, suspended in chromatin digestion buffer and stored (-80°C). AseI (100U, New England Biolabs, 37°C, 2 h). A ligation control (sees below and Supplementary Table S8) was added to the AseI digested chromatin, samples were diluted (~20-fold) and ligated with T4 DNA ligase (20U, Invitrogen). Following ligation, cross-links, protein and RNA were removed. pUC19 plasmid (27.4pg/2ml) was added as a sequence library preparation ligation control before phenol:chloroform (1:1) extraction and column purification.
attributes.tag: GEO Accession
attributes.value: GSM1262377