title: GSM1262385: G2_phase_synchronized_RNA-seq biological replicate #2; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: For RNA extraction, cells were harvested from 12 ml of each synchronized cell culture prior to cross-linking (4,000 rpm, 2 min, RT), washed with 5 ml of AE buffer (50 mM Sodium Acetate, 10 mM EDTA, pH 5.3), and suspended in AE buffer (400 μl). Cell suspensions were transferred to tubes containing an equal volume of phenol/chloroform/isoamyl alcohol (24/24/1 Ambion) and 400 μl of acid washed glass beads (Invitrogen). Tubes were transferred to a chilled block (-20°C) in a Geno/Grinder (SPEX sample prep 2010) and the cells were lysed (1,750 rpm, 8 x 30 sec on 60 sec off). Lysis was completed by freeze (-80oC, ~15 min), thaw before centrifugation (14,500 rpm, 5 min, 4°C). The aqueous phase was extracted three times with an equal volume of phenol/chloroform/isoamyl alcohol. RNA was isolated by precipitation with 1/10th volume ammonium acetate (5 M) and 2 volumes of 100% ethanol at -80°C (>30 min/over-night) before centrifugation (14,500 rpm, 10 min, 4°C). RNA was washed with 70% ethanol (350 μl; 14,500 rpm, 5 min, 4°C) and air dried (37°C, ~15 min). RNA pellets were suspended in RNASecure (80 μl; Ambion) and dissolved by heating (60°C, 10 min).
attributes.tag: GEO Accession
attributes.value: GSM1262385