Transcriptome Profiling of Fish Tissues

Illumina sequencing was performed using three tissues (the intestine, brain, and muscle) obtained from three fish in each group. RNA was extracted as previously described and enriched by oligo(dT) beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized by reverse transcriptase; RNA was then digested by RNaseH, and second strand cDNA was synthesized by DNA polymerase I system. After purification and repair, poly(A) was added to double-stranded cDNA and sequencing adapters were connected. AMPure XP beads were used to screen cDNA (250–300 bp) for PCR augmentation. After secondary purification, the library was constructed and quantified by qRT-PCR (≥ 2 nM), followed by sequencing on Illumina HiSeq 4000. Sequencing was performed by Novogene Co. (Beijing, China). Clean reads (> 100 bp) were obtained by removing reads containing adapters or poly-Ns from the raw data. Clean reads were mapped to the reference genome of C. idella with HISAT2 [58 (link)].