GSM9890586: no-Ag control, biological replicate 5; Pseudomonas aeruginosa PAO1; RNA-Seq
RNA was extracted from the membrane filters using the Qiagen RNeasy Kit with modifications to the manufacturer's protocol. Briefly, the tubes containing the filters were thawed on ice for 20 minutes, followed by the addition of 100 µL of 15 mg/mL lysozyme in Tris-EDTA (TE) buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). The tubes were vortexed at maximum speed for 10 minutes and then placed on ice for five minutes. Next, 350 µL of buffer RTL were added to each tube; the tubes were briefly vortexed and then centrifuged for 10 seconds at 5,000 xg. The supernatant was transferred to the Lysing Matrix E bead tubes (MP Biomedicals) and homogenized using a FastPrep-25 Classic homogenizer (MP Biomedicals) using three rounds of 50 seconds, with 5 minutes of cooling the tubes on ice between each round. The tubes were centrifuged at 17,000 xg, and the supernatant was transferred to new microcentrifuge tubes. Next, 220 µL of absolute ethanol was added, and the tubes were inverted five times to mix. The RNA was then captured on the spin columns from the kit per manufacturer's instructions and eluted into 20 µL of RNase-free water. Extracted RNA was stored at -80°C for sequencing. Ribosomal RNA depletion was performed using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina), and quality was assessed using a Bioanalyzer 2100 (Agilent). Library construction was performed using the TruSeq Stranded Total RNA kit (Illumina).