title: Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.
design: Library preparation and RNAseq sequencing were performed at Zymo Research Inc. (Irvine, CA), where each RNA sample (260 ng) was purified using Zymo-Seq RiboFree Total RNA Library kit (Zymo Research). Ribosomal RNA was removed from RNA samples, which were then 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 min; 16 cycles of denaturation at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 60 sec; and final extension at 72 C for 7 min. Successful library construction was confirmed with Agilents D1000 ScreenTape Assay on TapeStation.