GSM8014395: 20µM-N-80/C-25_1; Pseudomonas aeruginosa PA14; RNA-Seq
RNA isolation from the cells was carried out using the RNeasy mini kit (74104, Qiagen). To begin, 700 ul of RLT buffer (from the kit) supplemented with 1% β-mercaptoethanol was added to the cells and vortexed for 30 sec. The mixture was then centrifuged at 8000xg for 2 min, and 700 ul of the resulting supernatant was carefully recovered and mixed thoroughly with 500 ul of ethanol by pipetting. Subsequently, 700 ul of the mixture was transferred to a mini kit column and centrifuged at 8000xg for 30 sec. The flowthrough was discarded, and 350 ul of RW1 buffer (from the kit) was added to the column prior to spinning at 8000xg for 30 sec. The flowthrough was once again removed, and 80 ul of Dnase I solution, which contained 10 ul of Rnase-free Dnase Set and 70 ul of buffer RDD (79254, Qiagen), was added to the column. The column was then incubated for 15 min at room temperature. Following this, 350 ul of RW1 buffer was added to the column prior to spinning at 8000xg for 30 sec. The column was then transferred to a new collection tube, and 500 ul of RPE buffer (supplemented with ethanol) was added to the column prior to spinning at 8000xg for 30 sec. The flowthrough was removed, and 500 ul of RPE buffer was added to the column prior to spinning at 8000xg for 2 min. The flowthrough was once again removed, and the column was dried by spinning at 8000xg for 1 min. Finally, the column was transferred to a new collection tube, and RNA was eluted with 30 ul and then 20 ul of DEPC-treated water each time for 1 min of spinning at 8000xg. The resulting RNA samples were stored at -80oC. The quality of the RNA samples was examined prior to sequencing by using gel electrophoresis, which revealed two sharp peaks with very low background . An RNA Integrity Number equivalent (RINe) of 8.4-9.2 and a concentration of approximately 40-60 ng/ul were obtained. Three independent RNA samples were sequenced for each experimental condition. Library construction and sequencing were executed at the Genome Analysis and Technology Core of the University of Virginia. Specifically, mRNA enrichment was conducted using the NEBNext® rRNA Depletion Kit (Bacteria) (E7850L, New England BioLabs). Transcriptome sequencing was then performed with 150-bp pair-end reads produced. The range of raw reads for each sample varied from 8-10 million, thus ensuring an optimal depth of sequencing.