RNA seq of orange-spotted grouper: Mixture of liver, spleen and kidney
Briefly, mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads, and then the purified mRNA was cleaved into small fragments using divalent cations under elevated temperature. First-strand cDNA was synthesized with random hexamer primer and M-MuLV Reverse Transcriptase. Second-strand cDNA was further synthetized and adapters were ligated to the products. cDNA fragments of preferentially 150-200bp were selected and purified. Then the size-selected and adaptor-ligated cDNA were subjected to PCR amplification for 15 cycles. At last, PCR products were purified and library quality was assessed on the Illumina HiSeq 2500 platform.