Illumina HiSeq 2000 sequencing; RNA-seq of Pseudomonas aeruginosa PAO1 wild-type and mutant containing a single nucleotide polymorphism in fusA1
RNA-seq of Pseudomonas aeruginosa PAO1 wild-type and mutant containing a single nucleotide polymorphism in fusA1
FusA1 mutant and progenitor P. aeruginosa PAO1 containing the pUCP20 empty vector, and fusA1 mutant containing pUCP20:fusA1 were grown in 50 mL M9 minimal media supplemented with 0.5% (w/v) glucose, 37oC with shaking (250 rpm) in 250 mL flasks. Samples were harvested at late exponential phase (OD600 of 0.6 – 0.8) where 1 mL of culture was added to an equal volume of RNAlater RNA stabilization solution. Samples were incubated for 15 min at 4oC and centrifuged at 21,000 x g for 20 min at 4oC. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and digested twice with on-the-column DNase I digestion, following manufacturer's guidelines. The concentration and purity of the RNA was measured using a NanoDrop ND-1000 spectrophotometer (Nanodrop technologies Inc, USA) and using agarose gel electrophoresis. Total RNA was sent to GATC Biotech (Germany) for rRNA depletion and RNA-sequencing. Nine indexed, strand-specific cDNA libraries were prepared, and samples were sequenced on an Illumina HiSeq 2000 with a 51-bp single-end read length (GATC Biotech, Germany).