a–c, Data from 513 control or reprogramming single cardiac fibroblasts and bulk RNA-seq data of neonatal cardiac fibroblasts and cardiomyocytes were analysed with PCA. To identify groups of related cells and genes, the top 400 genes with highest weight in the first three principal components were then analysed by unsupervised hierarchical clustering (a) and PCA (b, c). Representative genes in each of the four gene clusters identified by hierarchical clustering are listed to the right of hierarchical clustering heat map. Notably, in addition to cardiomyocyte, fibroblast and cell-cycle genes, immune response genes were identified as the other major gene cluster. b, PCA loading plot showing four major gene clusters. c, PCA score plot. Both hierarchical clustering and PCA results showed that bulk cardiac fibroblast and cardiomyocyte data were very close in distance and both of them were clustered together with single cells expressing high levels of immune genes. The dashed line separates immune-like single cells and other single cells. d, Markers of the major immune cell lineages. e, Violin plots of the expression of major immune cell lineage markers in bulk cardiac fibroblasts, bulk cardiomyocytes, immune-like single cells and other single cells. f, Expression of the macrophage marker Cd14 and the dendritic cell marker Cd11c (also known as Itgax) in each immune-like cell showed that 42 cells express macrophage markers and 3 express dendritic cell markers, with 4 cells expressing both. These data suggest that the immune-like cells are probably cardiac resident immune cells38, which also express cardiac fibroblast markers, such as Thy1. Although follow-up work to delineate the potential of these immune cells to be reprogrammed into iCMs will be of great interest, it is not the focus of this study. Therefore, for all subsequent analyses of the single-cell data, we focused on the non-immune cardiac fibroblasts.