RNA viral metagenome from Coxsackie virus infected mouse brain tissue II
Viral particles were purified and viral RNA was extracted using Trizol-LS. RNA was reverse transcript and amplified using the Transplex Whole Transcriptome Amplification kit (Sigma-Aldrich, St Louis, MO). WTA-amplified cDNA libraries were then used for the Genome Sequencer FLX systems sequencing library preparation. Double-stranded cDNA was treated as sonicated DNA and proceeded directly to fragment size selection using the titrated amount of Agencourt AMPure SPRI beads. The ends of the fragments were polished and ligated with 454 adaptors prior to emulsion PCR as recommended by the manufacturer's protocol. The 454 multiplex adaptors were generated according to the manufacturer's protocol and used for all libraries. The amplified material was sequenced in-house with the Genome Sequencer FLX pyrosequencing system (Roche, Branford, CT) using the Titanium chemistry according to the manufacturer's protocol.