dietary protein level of Opsariichthys bidens
Total RNA was extracted by RNAiso Plus reagent. The purity and quantity of RNA were examined by using the BioTek Synergy 2 Multi-detection Microplate Reader and by 1.5% agarose gel electrophoresis. The mRNA with Poly(A) which was enriched by oligo (DT) magnetic beads was randomly broken into short fragments by adding fragmentation buffer, using a short fragment as a template to synthesize a strand of cDNA by reverse transcription. When the second strand was synthesized, dTTP in dNTPs were replaced by dUTP and then repaired cDNA terminal, added of A tail, connected UMI tag and sequencing adapter. The second strand of cDNA containing U was degraded by USER enzyme, and finally amplified by PCR to obtain the strand specific library. Illumina was used for sequencing after the library was qualified and the sequencing read length was PE150. Qubit2.0 Fluorometer was used for preliminary quantification and detect the insert size of mRNA-Seq library by Agilent 2100 Bioanalyzer