Creation of dual EMCV-IRES-integrated dengue virus
each RNA sample (260 ng) was purified using Zymo-Seq RiboFree Total RNA Library kit (Zymo Research). The purified, rRNA-depleted RNA samples were reverse-transcribed to cDNAs. The produced cDNAs were ligated with P7 adaptor sequence at 3 end, followed by second strand synthesis and P5 adaptor ligation to the opposite sites of the double stranded DNAs. After purification by DNA size (300-600bp) with beads in the kit, index PCR was performed (initial denaturation at 95 C for 10 mins, followed by 16 cycles of denaturation at 95 C for 30 secs, annealing at 60 C for 30 secs, and extension at 72 C for 60 secs, in addition to a final extension at 72 C for 7 mins). Successful library construction was confirmed with Agilents D1000 Screen Tape Assay on TapeStation. Libraries were sequenced on an Illumina Novaseq to a sequence depth of >30 million read pairs (150 bp paired-end sequencing).