RNA-Seq of Oenanthe oenanthe Liver
The mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads, fragmented ; bands in between 100-700 bp (main 200-300 bp) were selected. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The cDNA was subjected to end-repair and then was 3 adenylated. Adapters were ligated to the ends of these 3 adenylated cDNA fragments and the products amplified by PCR. The amplicons were purified with Agencourt AMPure XP beads (paramagnetic bead-based technology). Library quality was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were denatured, circularized and purified; the single strand circle DNA (ssCir DNA) were formatted as the final library product for sequencing. ssCir DNA library was amplified with phi29 to make DNA nanoball (DNB) which generate around 300 copies of one single molecule. The DNBs were load into the patterned nanoarray and pair-end reads of ~100 bases were generated by BGISEQ-500 sequencing system.
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