2.3. RNA-sequencing and analysis

Bacteria were grown on TSA overnight, then colonies inoculated into TSB and cultured in a shaking incubator (16 h, 37 °C). The following day, the TSB culture was sub-cultured in fresh TSB and incubated for 2 h at 37 °C until reaching an absorbance of ~0.4 at 650 nm. Bacteria were centrifuged at ~ 5,000 × g for 5 min and the bacterial pellet reconstituted in 2 ml of tear fluid or PBS control and incubated for 5 h at 37 °C with agitation. The 5 h time point for was chosen for initial analysis to allow sufficient time for P. aeruginosa to express tear-associated changes in gene expression. The samples were then centrifuged at 5,000 × g for 5 min to obtain a bacterial pellet for RNA extraction. RNA was extracted from samples in each condition according to the protocol in RNeasy kit (Qiagen) including on-column DNase steps to remove any contaminating genomic DNA. The RNA was first checked with a nanodrop to assess concentration, and the quality evaluated using the Agilent Model 2100 bioanalyzer (RIN > 8) before proceeding with RNA-sequencing. The rRNA was then depleted using Ribozero kit from Epicenter (rRNA content < 10 %), this rRNA-depleted RNA was then used to generate the libraries according to the RNA-Sequencing Library Construction Kit (Ambion). Sequencing of the libraries was performed using a HiSeq 2000 (Illumina). RNA sequencing involved one replicate per condition, and sequencing quality, significance and differential gene expression determined using Rockhopper [25,26].

Gene set enrichment analysis on differentially-expressed genes was performed using ADAGE [27,28]. Enriched networks were combined with RNA-sequencing fold-change information via Cytoscape (https://cytoscape.org), then plotted within an untargeted interactome. ADAGE is a neural network model which identifies gene signatures associated with various biological processes. Each gene holds a weight value associated with each node, represented by edge strength. Gene signatures are determined from gene sets that contribute the highest positive and negative weights to a given node. Thus, both a positive and negative gene signature are produced from a single node [27,28].