GSM8330299: 30min_pi_39_Jvpna-1258_PHIKZ186; Pseudomonas aeruginosa PAO1; RNA-Seq
RNA was isolated from 200 µl of cells using the RNAsnap procedure (Stead et al. 2012). Two volumes of RNAprotect (Qiagen) were added and cells were incubated for 5 min. Cells were pelleted at full-speed for 20 min at 4 °C and the supernatant was removed. The pellet was resuspended in 100 µl SNAP buffer (0.025% SDS, 18 mM EDTA, 1% β-mercaptoethanol, 95% formamide (RNA-grade)). Samples were incubated for 7 min at 95 °C, cell debris was pelleted at full-speed for 5 min at room-temperature, and the supernatant was transferred to a new tube. 1.5 volumes of ethanol was added to the supernatant and the sample was mixed by pipetting. The sample was loaded onto a miRNeasy mini column (Qiagen) two times and spun at full-speed for 20 s at room-temperature. Columns were washed two times with 500 µl RPE buffer (Qiagen) and spun at 8k×g for one minute at room-temperature. One final spin was used to dry the column in an empty tube. 30 µl RNase-free water was added to the column and the RNA eluted at 8k×g for one minute at room-temperature. The elution was repeated with the flow-through to recover more RNA. The RNA concentration was determined by absorption at 260 nm. RNA was stored at -80 °C. RNA-sequencing was performed at the CoreUnit SysMed at the University of Würzburg. DNA was digested with DNaseI and the rRNA was depleted with the Lexogen RiboCOP META depletion kit. RNA library was prepared with the CORALL Total RNA-Seq Library Prep Kit V1 (Lexogen).