Fig. 4

A milk-enriched granulocyte gene signature indicates transcriptional dyanmics of the granulocyte-specific, localized immune response to mastitis. (A) Phylogenetic tree indicating the transcriptional relatedness of granulocyte clusters recovered via scRNA-seq. The red dotted line indicates high-level segregation of hierarchical clustering into nodes referenced in the results. (B) Pie charts showing the proportion of cells (pie slices) comprising the cells within each node identified in (A). Fill color of pie slices corresponds to sample identifiers. Pie size is proportional to 100% of cells comprising a node and is not proportional to the total absolute number of cells in each node. (C) Dot plot of signature genes (x-axis) and their expression patterns across granulocyte clusters (y-axis). Signature genes were identified for node 1 identified in (A). The full node 1 gene set (210 genes) is not shown and can instead be found in Supplementary Fig. 2. Only genes with log2FC > 1 for both clusters (c30, c46) are shown in this figure. Dot size within the plot indicates the percentage of cells in a cluster expressing a gene. Dot fill color indicates the relative expression level of a gene in a cluster. The phylogenetic tree on the left shows relatedness of granulocyte clusters, identical to as shown in (A). (D) Left: Stacked bar plot showing the percentage of cells (x-axis) comprising each granulocyte cluster used for scRNA-seq (y-axis). Fill color of bars corresponds to cells derived from blood (red) or milk (blue) samples. Bar size is proportional to 100% of cells comprising a cluster and is not proportional to the total absolute number of cells in each cluster. Clusters on the y-axis are listed (top to bottom) in ascending order of the percentage of milk cells comprising each cluster. Right: heatmap showing the average gene set enrichment score for the 210-gene node 1 gene signature. Fill color corresponds to the average gene set enrichment score for each granulocyte cluster. (E) Violin plots showing gene set enrichment scores (y-axis) for the 210-gene node 1 gene signature. Granulocyte clusters (x-axis) are separated into cells derived from blood samples (red) or milk samples (blue). c60 does not have a violin plotted for blood since no blood-derived cells were identified in the cluster. Individual points within each violin represent single cells. (F) Cytoscape Gene Ontology (GO) term enrichment and clustering analysis of the 210-gene node 1 gene signature. scRNA-seq single-cell RNA sequencing.