RNA-Seq of yellow drum: testis
Using the characteristic structure of polyadenylated tails found in the majority of eukaryotic mRNA, mRNA enriched with polyA tails was isolated using Oligo(dT) magnetic beads. Subsequently, the obtained mRNA was randomly fragmented with divalent cations. Fragmented mRNA served as templates for first-strand cDNA synthesis using random oligonucleotides as primers in the presence of 1st Strand Enzyme reverse transcriptase. The second strand of cDNA was synthesized using 2nd Strand Enzyme and dNTPs. Purified double-stranded cDNA underwent end repair, A-tailing, and adapter ligation, followed by selection of cDNA fragments approximately 200-316 bp in length. PCR amplification and purification of PCR products yielded the final library.