RNA-seq of muscle, liver, gill, heart, kidney, swim bladder and sexual gland : female
First-strand cDNA synthesis was performed by combined the 17l of fragmented mRNA with 6l of 1st Strand Buffer and 2l of Strand Enzyme Mix and incubated at 25C for 10 minutes, 42C for 15 minutes and 70C for 15 minutes. For second strand synthesis, we added 20l of 2nd Strand Buffer and 5l of Strand Enzyme Mix into 25l of 1st Strand cDNA and incubated at 16C for 60 min. Then, double-stranded cDNA was purified by using 90l of VAHTSTM DNA Clean Beads (1.8 ). Subsequently, End Prep Mix, dA-Tailing Buffer Mix and RNA Adapter were applied to end repair, A-tailing and adapter ligation of the double-stranded cDNA, respectively. The linked product was purified by using 40l of VAHTSTM DNA Clean Beads (1 ) and the fragment size was selected by using 80l of VAHTSTM DNA Clean Beads (0.7 ). For the cDNA fragments enrich reaction, we added 5l of PCR Primer Mix and 25l of Amplification Mix 1 into 20l of purified product and ran the initial denatured at 98C for 30 sec, followed by 15 cycles of denatured at 98C for 10 sec, annealed at 60C for 30 sec, and extended at 72C for 30 sec, and finished with a final extended at 72C for 5 min. RNA-sequencing libraries were quantified using 8 polyacrylamide gel. Then the library was sequenced on the Illumina HiSeqTM 2500 platform and 150bp paired-end reads were generated.