RiboSeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics

Vero cells were infected with the different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics at MOI:3 to assess their viral transcriptome. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.