Illumina HiSeq 2000 sequencing; Pseudmonas aeruginosa PAO1 wild-type and delta prpC cultured on MOPS Succinate and MOPS Succinate + Propionate
Pseudmonas aeruginosa PAO1 wild-type and delta prpC cultured on MOPS Succinate and MOPS Succinate + Propionate
Pseudomonas aeruginosa strain PAO1 (wild-ype and prpC) was grown in 40 ml MOPS with succinate as the sole carbon source, 37 °C with shaking (250 rpm) in baffled flasks (500 ml). During exponential growth, 500 uM propionate was added to the cultures (H2O added to controls). Samples were harvested during mid-exponential growth. Four conditions, 12 samples total (triplicate). 5 ml of culture was harvested from each sample and 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 bacterial Ribo-Zero rRNA Removal Kit (Illumina). The integrity of the RNA was evaluated using an RNA 6000 Nano LabChip and an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany). Eight 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). 5 ml of culture was harvested from each sample and 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 bacterial Ribo-Zero rRNA Removal Kit (Illumina). The integrity of the RNA was evaluated using an RNA 6000 Nano LabChip and an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany).  12 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).