RNA-seq of Danio rerio larvae
To analyse the transcriptome response to NNV in zebrafish larvae infected via the brain, samples from uninfected and infected larvae were collected at 1, 3 and 5 days post-infection for high-throughput transcriptome sequencing. Whole larvae were harvested under RNAse-free conditions in pools of 4 larvae each (3 biological replicates/4 larvae per replicate). Total RNA was isolated using the Maxwell RSC simplyRNA Tissue kit (Promega) in accordance with the manufacturers instructions.The RNA concentration and purity were measured with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., USA), and RNA integrity was analysed in an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA) according to the manufacturers instructions. All samples showed an RNA integrity number (RIN) over 8.0 and were used for Illumina library preparation. Double-stranded cDNA libraries were constructed using the TruSeq Stranded mRNA Kit Sample Prep Kit (Illumina, USA), and sequencing was performed using Illumina NovaSeq 6000 technology at Macrogen Inc., Korea (Republic of Korea).