sequencing the transcriptome of tinca tinca
First of all, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Then, fragmentation of mRNA was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). After that, random hexamer primer was used to reverse-transcribe the mRNA fragments and cDNA fragments of preferentially 150~200bp in length were selected. After PCR amplification and purification, quality of the cDNA library was assessed on an Agilent Bioanalyzer 2100 system. Sequencing was performed on an Illumina Hiseq 2000 platform and millions of 125bp paired-end reads were generated.