2.10 Analysis of stress response genes
To reveal RNA expression changes in response to stress, we acclimated 100 adult shrimp for seven days at 28 °C in oxygenated water in individual 120 L buckets. Shrimp were fed with clams at 5% of the total shrimp weight daily. Every day, 50% of the water was changed. To evaluate the transcriptional response of the shrimp to cold stress, 20 shrimp were maintained at 28 °C (CT, controls) and 20 shrimps were maintained at 10 °C (LT) chiller, and the eyestalks from both groups were sampled for RNA extraction at 12 h and 48 h, at which time the shrimps began to die according a pilot experiment conducted under the same conditions. Eyestalk mRNA was subjected to transcriptome sequencing using the Illumina Hiseq X Ten system (carried out by Novogene Biotechnology, Tianjin, China). We filtered the raw data to remove those reads containing ambiguous bases (‘N’) or large numbers of low-quality positions (> 10 positions with quality scores < 10). Hisat2 (v2.0.4) (Kim et al., 2015) was then used to map the high quality reads to the M. japonicus genome. Read counts were calculated using HTseq software (Anders et al., 2015) and the normalized read counts (fragments per kilobase of transcript per million mapped reads; FPKM) was used to estimate the expression level of the genes. Differentially expressed genes (DEGs) were identified using DESeq2 (Love et al., 2014) and genes with a log2 (fold-change (FC)) ≥1 and adjustedp -value <0.05 were defined as significant DEGs.