GSM7105079: PAO1_empty_I_1; Pseudomonas aeruginosa; RNA-Seq
At OD (600 nm) = 2, 2 mL cells was harvested, 20 % ice cold stop mix solution (95 % v/v ethanol, 5 % v/v phenol, saturated pH 4.5) was added and the mixture was immediately snap-frozen in liquid nitrogen. Next, samples were thawed on ice and their pellets were collected (20 min, 4 °C, 4500 g) for resuspension in 300 µL of a 0.5 mg/mL lysozyme solution in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Subsequently, samples were treated via the hot phenol method, followed by ethanol precipitation to extract the RNA. Next, the samples were treated with DNase I and the RNA was further purified using ethanol purification. RNA samples were converted into cDNA using the Illumina Stranded Total RNA Prep Ligation Ribo Zero Plus kit. The Bioanalyzer and the DNA high sensitivity kit (Aligent) were used to verify rRNA depletion and evaluate the average fragment length, aiming for fragments around 500 bp. All samples (n = 3 per condition) were pooled together in equimolar amounts to obtain a 25 µL sample of 4 nM and the cDNA samples were paired-end sequenced via Illumina next-generation sequencing (Novaseq 6000)