Illumina iSeq 100 paired end sequencing; Transcriptome profiling by RNA-seq of the Pseudomonas aeruginosa strain PA14 deleted for the small RNA GssA compared with PA14 wild-type
Transcriptome profiling by RNA-seq of the Pseudomonas aeruginosa strain PA14 deleted for the small RNA GssA compared with PA14 wild-type
Total RNA for RNA-seq was extracted from PA14 wt and PA14ΔgssA grown in BHI medium at 37 °C with shaking until OD600 of 2. For each strain, three independent biological replicates were performed. Total RNA was prepared from pelleted bacterial cells resuspended in RNAprotect Cell Reagent (Qiagen), incubated for 5 min at room temperature, pelleted by centrifugation, and stored at −80°C until use. Total RNA extraction was performed by the RNeasy Mini Kits (Qiagen) according to the manufacturer's instructions, including RNase-free DNaseI in-column treatment. The concentration and quality of the extracted RNAs were assessed by a Biospectrometer (Eppendorf). RNA integrity was checked by electrophoresis of samples on denaturing (7 M urea) 6% polyacrylamide gel (dPAGE).  The concentration and the purity of the RNA starting material were determined spectrophotometrically while RNA Integrity Number (RIN) was measured on an Agilent TapeStation 4200. An additional DNase step was included to cleave DNA that could interfere with rRNA removal and negatively affect library preparation and sequencing. RNA libraries were generated starting from 3 μg of total RNA. rRNA was depleted with Illumina Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria) and libraries were generated according to Illumina TruSeq® Stranded mRNA Library Prep protocol. RNA libraries were accurately quantified with a fluorometric method: QUBIT dsDNA HS assay kit and the size and purity were verified on an Agilent TapeStation 4200.