Illumina NovaSeq 6000 paired end sequencing; Pseudomonas aeruginosa PA14 exposed to the aminothiol cysteamine during growth in synthetic cystic fibrosis sputum media (SCFM2)
Pseudomonas aeruginosa PA14 exposed to the aminothiol cysteamine during growth in synthetic cystic fibrosis sputum media (SCFM2)
Pseudomonas aeruginosa strain PA14 was grown in 25 ml SCFM2, 37 °C with shaking (200 rpm) in conical flasks (250 ml). During mid-exponential growth, freshly prepared cysteamine (100 µg/ml final concentration) was added to the cultures (H2O added to the controls). Samples were harvested after 4 h exposure, corresponding to the early stationary phase growth - six samples total (triplicate). 5 ml of culture was harvested from each sample and this was added to an equal volume of RNAlater® RNA stabilization solution. Samples were then centrifuged at 4000 rpm for 15 min (4 °C) and the pellets were stored at – 80 °C.  RNA was extracted using a RNAeasy Mini-Kit according to the manufacturers instructions. RNA was estimated with a NanoDrop ND-1000 spectrophotometer (NanoDrop technologies Inc, USA) and using agarose gel electrophoresis. Ribosomal RNA (rRNA) was then depleted from each sample (5 µg each) using the RiboMinus™ Transcriptome Isolation Kit, bacteria (Thermo Fisher Scientific). The integrity of the RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany).  Six indexed, strand-specific cDNA libraries were prepared from the above depleted RNA samples using the TruSeq RNA Sample Prep Kit (Illumina).