GSM6705123: P. aeruginosa, cells with 2 chromosomes, replicate 3; Pseudomonas aeruginosa PA14; RNA-Seq
For RNA protect: 1 mL of culture was mixed with the same volume RNAprotect™ Bacteria Reagent incubated for 10 min and centrifuged. The pellets were flash-frozen and stored at -70°C. RNA extraction was carried out with the RNeasy Plus Kit in combination with QIAshredder™ columns. Treatment with DNase I was performed in solution. For FACS: Key to successful RNA recovery is the gentle formaldehyde fixation at 4˚C. Aliquots of fixed samples were adjusted to approx. 1.8*107 cells/mL in 30 ml volume each and stained with SYBR Green. Sorting of 5.4*108 cells based on the FITC-signal directly into RNAprotect was performed with the BD FACSAria Fusion. The sorted cells were collected on a filter from which RNA was extracted using a combination of Lysozyme and Proteinase K digestion with bead beating, and purified with NucleoZOL. Ribosomal RNA depletion was performed with the NEBNext Bacteria kit. The libraries were prepared with the Illumina TruSeq kit