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The opportunistic pathogen Pseudomonas aeruginosa controls the production of virulence factors via its quorum sensing (QS) systems. The addition of 100 M 4-GB to succinate-containing medium led to a strong reduction of pyocyanin production in strain PAO1gbuA, whereas pyocyanin production by the wildtype was not affected. To investigate possible transcriptional differences between the two strains RNAseq analysis of strains PAO1 and PAO1gbuA in the presence of 4-GB was performed. RNA was extracted in late exponential phase. For the isolation of RNA for RNAseq analysis, pre-cultures of strains PAO1 and PAO1gbuA were grown in medium B containing 20 mM succinate for 12 h at 37C. Cells were washed and used to inoculate main cultures in medium B containing 20 mM succinate and 100 M 4-GB in triplicates with an OD600 of 0.001. After incubation for 11 h at 37C, the respective triplicates were combined, harvested by centrifugation and resuspended in medium B without complementing solutions. From these cell suspensions, RNA was extracted with the innuPREP RNA Minikit from Analytik Jena (Jena, Germany) according to the manufacturers instructions. Residual DNA was removed by digestion with 10 U RNase-free DNase I (Thermo scientific) for 1 h in the presence of RiboLock RNase inhibitor (Thermo scientific). After DNA digestion, the RNA was again purified with the same kit. RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). Ribosomal RNA molecules were removed from total RNA with the Ribo-Zero rRNA Removal Kit (Illumina, San Diega, USA) and removal of rRNA was checked with the Agilent RNA Pico 6000 Kit on an Agilent 2100 Bioanalyzer. cDNA libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA), and the resulting cDNA was sequenced paired end on an Illumina MiSeq system using 75 bp read length.

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