GSM8430327: Trm2KO no hygB #1; Saccharomyces cerevisiae; RNA-Seq
The 200 mL cell pellet was resuspended in 8 mL of lysis buffer (60 mM sodium acetate pH 5.5, 8.4 mM EDTA) and 800 μL of 10% SDS. One volume (8.8 mL) of phenol was added and vigorously vortexed. The mixture was incubated at 65℃ for five minutes and was again vigorously vortexed. The incubation at 65℃ and vortexing was repeated once. Then, the mixture was rapidly chilled in an ethanol/dry ice bath and centrifuged for 15 minutes at 15,000 x g. The total RNA was extracted from the upper aqueous phase using a standard acid phenol-chloroform extraction. The extracted total RNA was treated with 140 U RNase-free DNase I (Roche, 10 U/μL) at 37℃ for 30 min. The DNase I was removed through an acid phenol-chloroform extraction. First, small RNA (tRNA and small rRNA) was diminished using the MEGAclear Transcription Clean-Up Kit (Invitrogen) to purify RNA >200nt. Then, two consecutive Dynabeads oligo-dT magnetic bead selections (Invitrogen, USA) were used to purify poly(A) RNAs from 140 μg of small RNA depleted RNA. The resulting RNA was ethanol precipitated and resuspended in 14 μL. Subsequently, we used the commercial riboPOOL rRNA depletion kit (siTOOLs Biotech, Germany) to remove residual 5S, 5.8S, 18S, and 28S rRNA.