RNA sequencing and gene analysis of unstimulated and stimulated MRSA cultures

Aim: the goal of this study is to compare Quorum sensing related gene expression in MRSA cultures incubated with an aza-derivative Methods: 1x10^6 CFU/ml bacteria were grown for 16 hours in the presence or absence of an aza-derivative (100 microM) or vehicle (DMSO 0,05%). The bacterial RNA was purified using the GRS Total RNA Kit – Bacteria (#GK16.0100, GRISP Research Solution) following the manufacturer’s instructions. Integrity was evaluated both by agarose gel electrophoresis and BioAnalyzer analysis before RNA-seq analysis was carried out. RNA-seq libraries were prepared with the TruSeq RNASample Prep kit (Illumina, San Diego, CA). The total RNA samples were subjected to poly(A) enrichment, and 100 nucleotides of sequence was determined from both ends of each cDNA fragment using the HiSeq platform (Illumina). Sequencing reads were annotated and aligned to the USA300 MRSA reference genome using TopHat. Comparisons were made between MRSA vehicle (DMSO 0,05%) and MRSA treated with the aza-derivative. Result: a set of genes known to be functionally related to agr QS system were selected a priori by retrieving all the genes that were used to construct a Boolean network describing quorum sensing in Staphylococcus aureus. The genes were divided into four categories: virulence, metabolism, membrane, and gene expression.