Illumina HiSeq 4000 sequencing; Bulk RNA Barcoding and sequencing (BRB-seq) of Mock, ICV, and IDV infected airway epithelial cell cultures of human, porcine, and bovine origin at 33°C and 37°C
Bulk RNA Barcoding and sequencing (BRB-seq) of Mock, ICV, and IDV infected airway epithelial cell cultures of human, porcine, and bovine origin at 33°C and 37°C
We performed a temporal transcriptome analysis on human, porcine, and bovine airway epithelial cell (AEC) cultures infected with ICV and IDV, as well as uninfected hAEC cultures, incubated either at 33°C or 37°C. AEC cultures from the different species were harvested at 24, 48 72, 96 hpi and processed for Bulk RNA Barcoding and sequencing (BRB-seq). Virus-infected and control hAEC cultures were lyzed with 350 uL of MR1 lysis buffer from NucleoMag RNA kit(Macherey-Nagel) according manufacturers guidelines, samples were stored at -80°C for later analysis. Cell lysates were collected at 24, 48, 72, and 96 hpi from cultures  incubated at 33°C and 37°C. Isolation and culturing of primary material was performed as previously described (Gultom, M., Laloli, L. & Dijkman, R. Well-Differentiated Primary Mammalian Airway Epithelial Cell Cultures. in Methods in Molecular Biology 2203, 119–134). Until the experimental procedures, all AEC cultures were maintained at 37°C in a humidified incubator with 5% CO2. Total cellular RNA from mock and virus-infected hAEC cultures was extracted with the NucleoMag RNA kit (Macherey-Nagel) according manufacturers guidelines on a Kingfisher Flex Purification system (Thermofisher). Total RNA concentration was quantified with QuantiFluor® RNA System (Promega) according manufactures guidelines on a Cytation 5 multimode reader (Biotek). BRB-seq protocol (Alpern, D., Gardeux, V., Russeil, J. et al. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome Biol 20, 71 (2019). https://doi.org/10.1186/s13059-019-1671-x)