Trunk of normal fish, replicate #2
One microgram total RNA from each sample was used as the beginning material for sequencing library construction. The NEBNext rRNA Depletion Kit was used for rRNA depletion. After rRNA depletion, Agencourt RNAClean XP Beads from Beckman Coulter were used for RNA purification. Then, the NEBNext Ultra Directional RNA Library Prep Kit for Illumina was used for RNA fragmentation, first and second strand cDNA synthesis, double-stranded cDNA end repair. Double strand cDNAs were purified using the Agencourt AMPure XP from Beckman Coulter and ligated to adaptors of NEBNext Multiplex Oligos for Illumina. Finally, the Q5 Hot Start HiFi PCR Master Mix was used for PCR enrichment of the adaptor-ligated DNA. The concentration and quality of the constructed sequencing libraries were measured by using the Agilent High Sensitivity DNA Kit and a Bioanalyzer 2100 from Agilent Technologies.