RiboSeq of BSR cells infected at MOI 3 with Theiler's murine encephalomyelitis virus, compared to mock-infected controls, harvested at 10 hpi

BSR cells were infected with the Cardiovirus B archetype, Theiler's murine encephalomyelitis virus (TMEV), and mutants thereof, and subjected to ribosome profiling to analyse the viral translatome, frameshifting, and ribosome pausing events. Samples were harvested at 10 hpi 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 19-35nt long. Fragments were cloned into adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5'-end of the 3'-adapter and the 3'-end of the 5'-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform.