title: RNA-seq of Pseudomonas aeruginosa: wspFpelpsl knockout Treated
design: Samples were centrifuged at 10.000 g, and were resuspended in 2 volumes of RNA protect Bacteria Reagent (Qiagen). After 5 min incubation at room temperature the cells were centrifuged at 10.000 g and the supernatant was discarded. The cells were frozen at -80oC until further analysis. Isolation of RNA was performed with RNeasy minipurification kit (Qiagen) with DNase I treatment according to the manufacturers guidelines. Subsequently, synthesis of cDNA was done from 1 g RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). Quality control of RNA samples for purity and integrity was done by NanoDrop spectrophotometer measurements (Thermo Scientific), Agilent 2100 Bioanalyzer (Agilent Technologies), and visual inspection of samples after agarose gel electrophoresis. RNA was purified from three replicate set of cultures for RNA sequencing and qRT-PCR. Assessment of global gene expression was done by Illumina RNA sequencing (RNA-seq). RNA-seq was conducted for three biological replicates of each sample. Ribosomal RNA was removed using the Ribopool 2 targeting P. aeruginosa rRNAs (SiTOOLs Biotech). Libraries were produced using the NEBNEXT Ultra II RNA Library Prep (New England Biolabs), and were sequenced using the Illumina HiSeq2500 platform with paired end protocol and 150nt read lengths.