title: GSM6032355: ARP42_T0_C; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Wild type and mutant strains were grown in a 200 ml liquid PMG+N medium using a shaking incubator (200 rpm at 30o C) to reach between 1.0x106 and 10x106 cells/ml. For each culture 100 ml was removed for the T0 timepoint, and the rest of the culture was washed with pre-warmed PMG-N and incubated for 24 hours in 500 ml of pre-warmed PMG-N using a shaking incubator (200 rpm at 30o C). For RNA extraction, cells were washed with ice-cold PBS and resuspended in 500 ¼l of ice-cold RNA extraction buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2% Triton X-100, 1% SDS, 100 mM NaCl). Then we added 500 µl of Phenol (acidic phenol pH 4.5, Sigma) and 500 µl of glass beads (acid washed, Sigma). The tubes were vortexed vigorously and incubated at 65¡C for 45-60 min. Next, the tube was placed on ice for 5 min. and centrifuged (1300 g, 5 min, 4¡C). The upper aqueous part was collected and transferred to a tube with 500 µl of chloroform (Sigma Aldrich), vortexed and centrifuged (1300 g, 5 min, 4 ¡C). The upper phase was collected and subjected to RNA precipitation at -20o C overnight. The precipitated RNA was washed once with 70% ethanol and dissolved in 30 ¼l H2O. To remove rRNA, 3 µg of purified total RNA was treated with Ribominus Eukaryote System v.2 kit (Ambion, Thermo Fisher Scientific). To generate sequencing libraries, a total of 100 ng of rRNA-depleted stocks and Illumina Stranded mRNA Prep Ligation kit (Illumina) were used. To quantify the samples, Qubit (HS dsDNA) was used, and samples were sequenced using an Illumina Nextseq 2000 platform (P3 100 cycle kit, 58 + 58 cycles, paired-end sequencing) at the BEA facility (Huddinge, Swe den) following the manufacturer's instruction.