Project description:To obtain a preliminary picture at the transcriptional level of the set of genes linked to the regulatory activity of the small RNA GssA of P. aeruginosa PA14, we analyzed the global transcription profile of PA14deltagssA strain in comparison with PA14 by an RNA-seq approach, extracting total RNA at OD600 of 2, corresponding to early stationary phase, when the two strains were grown in Brain Heart Infusion (BHI) rich medium, a condition in which, at the time of its identification, a relevant expression of GssA was noted. For each strain, three independent biological replicates were performed. 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. RNA libraries were then sequenced on an Illumina HiSeq with paired-end reads 150 bp long. For RNA-Seq data analysis, P. aeruginosa UCBPP-PA14 (NCBI: NC_008463.1) genome and annotation (GTF) were downloaded from the Pseudomonas Genome Database. Alignment of paired Fastq RNA-Seq reads to P. aeruginosa UCBPP-PA14 genome was performed using the STAR aligner (v2.7.0) with the quantMode TranscriptomeSAM GeneCounts option turned on. SAM files were converted to BAM, sorted, and indexed using SAMtools. Differential gene expression analysis was performed using DESeq2 using a design = ~ condition model and dedicated R scripts. The false discovery rate was controlled using the Benjamini-Hochberg procedure, with an adjusted p-value (FDR) ≤ 0.1. P. aeruginosa gene IDs were annotated with a gene description field using a custom tool (IdToGeneNamesScript) using the P. aeruginosa UCBPP-PA14 GTF file as annotation input.
