Organoids were collected, and single-cell suspensions were made using a papain dissociation system (Worthington Biochemical Corp.). Single-cell suspensions were loaded onto the Chromium Controller (10x Genomics) for droplet formation. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ Reagent Kit (10x Genomics). Samples were sequenced on the NovaSeq.

R v3.4.2 (R Core Team) was used for all statistical analysis. Sequencing results were demultiplexed into Fastq files using Cell Ranger (v3.0.2; 10x Genomics) mkfastq function. Samples were aligned to GRCh38 (10x Genome). The count matrix was generated using the count function with default settings. Matrices were loaded into Seurat v3.1.5 for downstream analysis. Cells with <200 unique molecular identifiers or high mitochondrial content were discarded. Using FindIntegrationAnchors and IntegrateData functions, all libraries were integrated into a single matrix. Principal component values that were statistically significant were identified, and a cutoff point was determined using the inflection point after using the PCElbowPlot function. Clusters were determined using the RunUMAP, FindNeighbors, and FindClusters functions on Seurat. Cells were considered infected if transcripts aligned to viral ORF1ab, Surface glycoprotein (S), ORF3a, Envelope protein (E), Membrane glycoprotein (M), ORF6, ORF7a, ORF8, Nucleocapsid phosphoprotein (N), or ORF10. DEGs from the FindMarkers function were used to perform PANTHER-GO statistical overrepresentation tests for up-regulated and down-regulated genes in each condition shown in Fig. 5 B. Gene lists for Fig. 5 C were obtained from gene set enrichment analysis databases (https://gsea-msigdb.org/gsea/index.jsp).

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