RNA extraction and library preparation for Illumina sequencing

Total RNA from livers was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quantity, purity and integrity were determined by 1% agarose gels and a Nanodrop 1000 spectrophotometer (NanoDrop, USA). High-quality RNA was selected for high-throughput sequencing, and samples from the three fish for each time point were combined. mRNA was purified from total RNA using oligo-dT-attached magnetic beads and fragmented using divalent cations under elevated temperature. First-strand cDNA was synthesised using random primers and reverse transcriptase. Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. A paired-end library was constructed using the Genomic Sample Prep Kit and sequenced using the Illumina Mi-Seq platform (Yin et al. 2016).