Template cDNAs were synthesized from RNA samples using SuperScript VILOTM cDNA synthesis kit (ThermoScientific, Waltham, MA). Resulting cDNAs were purified using the QIAquick 96 PCR Purification Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA). These purified cDNAs were used in the TaqMan qPCR validation assay. cDNAs were analyzed by qPCR using the reference gene TAF11 primers and probe in duplex with primers and probe for each select gene. Linear gene-to-reference gene ratios were calculated for each gene and sample. Normalized microarray values were used to calculate gene-to-reference ratios for each sample and gene. Spearman correlation (GraphPad Software, La Jolla, CA) was calculated between qPCR and microarray values. The list of primers is provided in Supplementary file 10 (‘WNV-ND transcriptome validation’).

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