GSM4011069: 65.0 fL_RNAseq; Saccharomyces cerevisiae; RNA-Seq
For each of the 24 samples, from ~3E+07 cells total RNA was prepared with the hot phenol method. Briefly, the frozen pellets were re-suspended in 0.4 ml TES buffer (10 mM Tris pH = 7.5, 10mM EDTA, 0.5% SDS), in DEPC-treated water, and ~0.05 ml glass beads were added. Then, 0.4 ml of acid phenol was added to each pellet, and the samples were incubated at 65 °C for 30 m, and vortexed briefly every 5 m during that time. The samples were centrifuged at 14,000 g for 5 m, and 0.3 ml of the top, aqueous layer were placed in a 2-ml screw-cap tube containing 1 ml cold ethanol with 40 μl of a 3M sodium acetate solution. The samples were incubated at 4 °C overnight and then centrifuged at 14,000 g for 20 m. The pellets were washed with 80% ethanol and centrifuged at 14,000 g for 5 m. The pellets were air-dried and resuspended in 25 μl of DEPC-treated water. For the RNAseq libraries, we used the same approach we had described (Blank et al., 2017; EMBO J. 2017 Feb 15;36(4):487-502. doi: 10.15252/embj.201695050), except that we did not select for polyA-tailed RNAs. Instead, from total RNA, we depleted rRNA, using the 'Ribo-Zero™ Magnetic Gold Kit (Yeast)', according to the manufacturer's instructions. RNA-Seq, the 24 libraries were loaded onto 2 lanes (denoted L005 and L006 in the file names)