Comprehensive RNA Extraction and Sequencing

Total cellular RNA from mock and virus-infected AEC cultures was extracted using the NucleoMag RNA kit (Macherey-Nagel) according to the manufacturer’s guidelines on a Kingfisher Flex Purification system (Thermo Fisher Scientific). Total RNA concentration was quantified with the QuantiFluor RNA System (Promega) according to the manufacturer’s guidelines on a Cytation 5 multimode reader (Biotek). The BRB-seq libraries were generated and sequenced as described previously to a depth of approximately 5 million raw reads per sample (Alithea genomics) (37 (link)). The host genome count matrices were generated by aligning the sequencing reads with STAR (v2.7.9a) against the corresponding reference genomes of Homo sapiens (GRch38.100), Sus scrofa (Sscrofa 11.1.100), or Bos taurus (ARS_UCD1.2.100). The viral count matrices were generated by aligning the sequencing reads with STAR (v2.7.9a) against a concatenation of the corresponding reference genomes of ICV (GenBank AF170573 – AF170576, AM410041 – AM410043) and IDV (GenBank KF425659 – KF425665). The resulting host and viral count matrices with unique molecule identifier (UMI) counts were subsequently used as input for data analysis.