NextSeq 500 paired end sequencing; RNA-Seq analysis of an industry isolated Pseudomonas aeruginosa strain following exposure to preservatives and product formulations.
RNA-Seq analysis of an industry isolated Pseudomonas aeruginosa strain following exposure to preservatives and product formulations.
In total 7 test conditions (termed 1-7) and one control condition (growth in liquid media) were included, each with 4 biological replicates. The results from the test conditions were compared to the control, to determine differentially expressed genes when the industrial strain was exposed to the preservatives and product formulations. Test conditions included:   1) The preservative benzisothiazolinone (BIT) at 20% of the minimum inhibitory concentration (MIC)  2) BIT at 50% of the MIC 3) Phenoxyethanol (POE) at 20% of the MIC 4) POE at 50% of the MIC  5) BIT and POE in combination, both at 20% of the MIC  6) Unpreserved laundry detergent (LD) 7) Laundry detergent (LD) in combination with BIT at 20% of the MIC. After exposure to the control and test conditions for 24 hours the cultures for each of the sample replicates were promptly snap-cooled in an ethanol and dry ice bath, before centrifuging at 20,000 x g at 4ºC for 1 minute. The supernatant was removed and pellets were immediately snap-frozen and stored at -80ºC until required. Total RNA was extracted within one week of harvesting cells using the RiboPure™ RNA Purification Bacteria Kit (Ambion, Life Technologies Ltd, UK) according to the manufacturer's instructions. A MICROBExpress™ bacterial mRNA enrichment kit (Ambion), was used to enrich for messenger RNA (mRNA) from the extracged total RNA.  Complementary DNA (cDNA) library preparations and sequencing were carried out in collaboration with the Genomics Research Hub at Cardiff School of Biosciences. The Illumina® TruSeq® Stranded mRNA Sample Preparation Kit was used to prepare sequencing libraries with the low sample protocol. The manufacturer's instructions describing the use of the protocol with previously isolated mRNA was followed; 5 μl of enriched mRNA within the Illumina recommended concentration range of 10 – 400 ng was added to 13 μl of the fragment, prime, finish mix to prepare sequencing libraries for each sample.