title: Corbicula fluminea Transcriptome Sequencing
design: Briefly, the poly-A containing mRNA molecules were purified from the 4µg of total RNA by  using poly-T oligo-attached magnetic beads using two rounds of purification. During the second  elution of the poly-ARNA, the RNA was fragmented using the divalent cations under 95°C. The  cleaved RNA fragments were reversely transcribed into first strand cDNA using random hexamers,  following by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA  fragments were performed end repair process to convert the overhangs into blunt ends, usingan End  Repair (ERP) mix. The 3’to 5’exonuclease activity of this mix removes the 3’overhangs and the  polymerase activity fills in the 5’ overhangs.A single ‘A’ nucleotide was then added to the 3’ ends of  the blunt fragments to prevent them fromligating to one another during the adapter ligation reaction.  A corresponding single ‘T’nucleotide on the 3’ end of the adapter provides a complementary  overhang for ligatingthe adapter to the fragment. This strategy ensures a low rate of chimera(concatenatedtemplate) formation.The multiple indexing adapters w ere ligatedto the ends of the  dscDNA, preparingthem for hybridization onto a flow cell. PCR was used to selectively enrich  those DNA fragments that have adaptermolecules on both ends and to amplify the amount of DNA  in the library. Thenumber of PCR cycles was minimized to avoid skewing the representation of  thelibrary.A gel purification procedure was carried out to select the fragments sized from 250 to  350bp to produce the library for cluster generation and sequencing. The library were qualified by  Agilent 2100 bioanalyzer and quantified by Qubit and qPCR. The cluster formation and sequencing  on the GAIIx platform were performed following the manufacturer’s standard cBot and sequencing  protocols.