RNA-Seq of Sparidentex hasta larvae: 6 h control_Replicate 3
The mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads and fragmented using fragmentation reagent. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and was 3 adenylated. Adapters were ligated to the ends of the 3 adenylated cDNA fragments followed by amplification. The PCR products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circular DNA (ssCir DNA) molecules were considered as the final library. The library was then amplified with phi29 to make DNA nanoball (DNB), which had more than 300 copies of one molecule. The DNBs were loaded into the patterned nanoarray paired-end reads were generated.