title: Illumina NovaSeq 6000 paired end sequencing; Pseudomonas aeruginosa PA14 exposed to the aminothiol cysteamine during growth in synthetic cystic fibrosis sputum media (SCFM2)
design: Pseudomonas aeruginosa PA14 exposed to the aminothiol cysteamine during growth in synthetic cystic fibrosis sputum media (SCFM2)
library_construction_protocol: 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).