GSM3244918: leo1D_T48_rep1; Schizosaccharomyces pombe; RNA-Seq
Cells were harvested at 3000 rpm at 25 °C. Then the cells were washed with phosphate buffered saline (PBS; pH 7.4) once. The pellet was re-suspended in 500 µl RNA extraction buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0)) and 500 µl acid phenol (pH 5.2) and then 500 µl acid-washed glass beads were added into the tubes. The tubes were vortexed vigorously at 4°C to disrupt cells. The tubes were centrifuged at 13,000 rpm for 5 min at 4°C and the upper aqueous phase was collected into a gel phase tube with same volume chloroform. After mixing the tubes were centrifuged at 13,000 rpm for 5 min at 25°C and the upper aqueous phase was collected. Ammonium acetate (pH 5.2) was added to a final concentration of 2.5 M along with 2.5 volumes of ethanol. The tubes were kept at −20°C for overnight for RNA precipitation. The precipitated RNA was washed with 70% ethanol once and dissolved in RNase free water. DNase I treatment was performed using TURBO DNase (Thermo Fisher Sci.) following the manufacturer's instructions. To remove rRNA, 4µg of purified total RNA was treated with ScriptSeq Complete Gold Kit Yeast (Epicentre). 40ng rRNA-depleted stocks were used to generate sequencing libraries using the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre).
GEO Accession
GSM3244918