Total RNA extracted from the different treatment conditions were analysed with the NanoString nCounter® analysis system [31 (link)], using a gene expression TagSet that targets 123 C.albicans genes—including three housekeeping genes, ACT1, LSC2 and THD3 [32 (link)]—and 60 C.elegans genes—including three housekeeping genes, rps-2, rps-4 and rps-23 [33 (link)]. The full list of genes with functions of the 183-genes can be found in Supplementary Table S1. Analyses of differential expression was performed using nCounter® with Elements™ XT Reagents according to manufacturer’s specifications. A multiplexed probe library (nCounter® elements CodeSet) was designed with two sequence-specific probes for each gene of interest. Probes were mixed with 100 ng of purified total RNA and allowed to hybridise (18 h, 67 °C). Samples were loaded on an nCounter® SPRINT™ Cartridge and processed with an nCounter® SPRINT™ Profiler (NanoString Technologies, USA) to quantify the transcripts. The nCounter raw expression data file (RCC) obtained was uploaded into the nSolver Analysis Software 4.0 for review of quality control metrics. The data were grouped between the experiments and control, and their expression ratios determined.
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