GSM6475307: T. maritima, 80, early exp, biol rep 5; Thermotoga maritima; RNA-Seq
Cell pellets were defrosted on ice and washed with 500 µL of DNase/RNase free water (New England Biolabs). Cells were resuspended in 700 µL of RLT buffer, and 50 mg of acid-washed glass beads (0.1 µm diameter) were added to a safe-lock tube containing the cell suspension. Cell disruption was performed with the OMNI bead mill Homogenizer (6.3 m/s) 2x. RNA was extracted with the RNeasy® Mini Kit (QIAGEN). The cells were harvested at early exponential, exponential, and stationary phases by centrifugation at 3500 rpm for 10 min at 4°C. The supernatant was discarded, and the remaining pellet was immediately frozen at -20°C for RNA extraction. 18 µl (2 µg) of total RNA was hybridized with the Pan-Prokaryote riboPOOL 3'-biotinylated probe (siTOOLs Biotech). The rRNA was subsequently depleted with Hydrophilic streptavidin magnetic beads (New England Biolabs). The depleted rRNA sample was purified before library preparation with RNA clean and concentration (Zymo research) and eluted in 15 µl of DNase-/RNase- Free Water. Samples were QC analyzed with the Agilent RNA 6000 Nano Chips and the Agilent 2100 Bioanalyzer system and stored at -80oC 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)