GSM6475278: T. ethanolicus, 45, stationary, biol rep 3; Thermoanaerobacter ethanolicus; RNA-Seq
Cells cleared from medium were snap-frozen in dry-ice and stored at -80oC until usage. For RNA extraction the frozen cell pellets were resuspended in 4 ml RNA-later (Invitrogen) while maintained on ice. A 200 µl sample was taken from the cell suspension for RNA isolation. The cells were incubated at room temperature for 5 min. The cells were then pelleted for 10 min at 4700xg. The cell pellet was resuspended in 100 µl TE buffer pH 7.5. 500 µl TRIzol reagent was added and mixed. The subsequent suspension was transferred to a pre-cooled 2 ml screw-cap tube supplemented with 250 µl sterile 0.1 mm and 1 mm glass beads. Cells were lysed by beating 6 rounds of 25 sec pulse at 6500 rpm and 5 min pause on watery ice. Ice-cold chloroform (200 µl) was subsequently added and mixed by shaking. Two phases were produced by centrifugation for 15 min. at 12,000xg. The transparent aqueous phase was transferred to a new RNAse-free pre-cooled tube without disturbing the interphase. Ethanol was added at 1 volume and mixed. The isolate was transferred to an RNAeasy column and centrifuged 15 seconds at 8000 xg to bind nucleic acids to the column. The column was rinsed with 350 µl RLT buffer by centrifugation. An 80 µl RNAse-free DNAse reaction mixture was added to the column and incubated 20 min at 30oC to digest DNA contaminants. The column was rinsed with 350 µl RW1, incubated 5 min and centrifuged. The column was then washed with 700 µl RPE, centrifuged and washed with 80% ethanol. The column was dried by a 2 min spin at 21.000 xg. To elute the RNA, 35 µl nuclease-free water was added and eluted by 1 min centrifugation at 8,000 xg. For rRNA depletion, 18 µl (1 µg) of total RNA was hybridized with the Pan-Prokaryote riboPOOL 3' biotinylated probe following the depletion protocol and stored at -80°C until library preparation. RNA libraries for RNA-seq were prepared using TruSeq RNA stranded kit following manufacturer's protocols and sequenced at the Utrecht Sequencing facility (USeq, The Netherlands)