The DEAD-box RNA helicases RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis

Purpose: We wanted to determine the transcriptome profile of PAO1 wild type, PAO1?rhlE1, PAO1?rhlE2 and PAO1?rhlE1?rhlE2 mutants on swarming conditions. Method: Cells were harvested using the RNA bacteria protect solution (QIAGEN). Total RNAs was extracted and purified using Monarch RNA isolation kit (NEB), treated with DNase I (Promega) three times to remove contaminating genomic DNA and re-purified again using phenol-chloroform. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina) . The samples were sequenced in single end mode on an Illumina HiSeq 4000 device and mRNA reads were trimmed and mapped to the NC_002516.2(PAO1) reference genome from NCBI using Stampy pipeline with defaut settings. Overall design: PAO1 wild type, PAO1?rhlE1, PAO1?rhlE2 and PAO1?rhlE1?rhlE2 mutants were grown in twelve 12-cm square swarming plates at 37°C. Samples from 6 plates were pooled and two replicates of each strain were therefore obtained. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Illumina). Then, libraries were prepared using the Illumina TruSeq stranded mRNA kit and validated on the Bioanalyzer 2100 (Agilent). Samples were sequenced using the Illumina HiSeq 2000, 100 bp single end read at the iGE3 genomics platform of the University of Geneva.