Figure 7: rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells.

A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa (A) and S. aureus (B) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa (C) and S. aureus (E). Copy numbers of P. aeruginosa (D) and S. aureus (F) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.