For metabolic RNA labelling of 293T transfected with a plasmid or Calu3 infected cells, growth medium was replaced with medium containing 4sU (T4509, Sigma) 24 h post-transfection and 3 hpi at a final concentration of 200 μM (a concentration that did not induce substantial cell cytotoxicity at 3 h labelling). Cells were collected with Tri-reagent at 2, 3, or 4 h after labelling for transfected cells or at 1, 2, or 3 h after labelling for infected cells (corresponding to 4, 5, and 6 hpi). RNA was extracted under reducing conditions and treated with iodoacetamide (A3221, Sigma) as previously described(Herzog et al., 2017 (link)). RNA-seq libraries were prepared and sequenced as described above.