title: GSM8397559: S. pombe cells, Rapamycin treated (100nM) - duplicate 2; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Yeast cells were harvested after 5 hours treatment with Rapamycin/Rapalink-1 (and untreated control), washed with DEPC water and pelleted. 750uL of TES and subsequently acidic phenol-chloroform was added, mixed and incubated at 65°C for 1 hour with repeated vortexing every 10 minutes for 10 seconds each. Samples were placed on ice for 1 minute, vortexed for 20 seconds and then centrifuged for 15 minutes at 4°C and 20,000 rcf. To pre-spun yellow phase-lock tubes, 700uL of acidic phenol-chloroform and 700uL of the water phase from samples were added and the tube was inverted multiple times to mix together, after which the tubes were centrifuged for 5 minutes at 4°C as previously described. To pre-spun phase-lock tubes, 700uL of chloroform:isoamyl alcohol (24:1) and 700uL of water phase from samples from yellow phase-lock tubes were mixed together and centrifuged at 4°C as before. To precipitate RNA, 500uL of water phase from the previous step were added to 2ml eppendorf tubes containing 1.5ml of 100% EtOH (cold) and 50uL of 3M NaAc pH 5.2, vortexed for 10 seconds and then incubated at -70°C for 30 minutes. The samples were then centrifuged again for 10 minutes at 20,000 rcf and 4°C. The supernatant was discarded and 500uL of 70% EtOH (in DEPC water at 4°C) was added and the tube was inverted to mix the sample. Majority of the supernatant was aspirated and the samples were spun down again briefly (5 seconds). The remaining superntant was removed and the pellet was allowed to air dry for 5 minutes at room temperature. Lastly, the pellet was resuspended in 100uL of DEPC water and incubated for 1 minute at 65°C. The pellet was then pipetted until dissolved and then gently vortexed for 10 seconds. RNA concentrations were measured using Qubit as per manufacturers' instructions. The samples were further processed by RNAeasy (Qiagen) cleanup with on column DNase treatments before double elution. RNA library formed by polyA capture and Illumina 150bp paired-end sequencing (Novogene, Cambridge)