Fig. 3

Single-cell RNA-sequencing identifies type I IFN-expressing cells early after activation. a The pDCs were coated with capture reagent, encapsulated in picoliter droplets, and stimulated individually with 50 µg/mL CpG-C for varying amounts of time. b After staining for surface marker expression and cytokine secretion, different pDC subsets were collected using fluorescence-activated cell sorting (FACS) and their transcriptomes were sequenced using the SORT-Seq protocol. c Heat map of the 774 cells that passed quality control filters representing transcriptome similarities as measured by Euclidean distance. The k-medoid clustering in combination with the raceID2 algorithm identified 8 distinct cell clusters. d t-SNE map showing all identified clusters. Different colors indicate clusters, different shapes indicate stimulation time. e The employed workflow allowed to link protein expression data acquired during FACS to the transcriptome data. The number of IFNα⁺ cells assigned to each cluster, and the percentage of sorted IFNα⁺ cells in each cluster, is plotted against the cluster name. f t-SNE map showing the fluorescence intensity of IFNα and TNFα as measured during FACS for each cell. g Shown are transcript counts for genes of the type I IFN response and the TNF gene in single cells stimulated for 2 h with CpG-C. IFNα⁺ cells, identified during FACS, are indicated in blue, other cells are shown in red. h Genes that were upregulated in cluster 5 compared to cells from all other clusters were detected (p < 10⁻⁸). Shown is the log2(fold change) for each gene. The color scale indicates the corresponding p value