We performed Seurat clustering analysis on 1524 stromal cells (DSC, FB1, and FB2) of P6D_10x sample and identified 1751 variable genes (FindVariableGenes). We used log-transformed gene expression matrix of variable genes to generate diffusion map by destiny R package. To identify genes on pseudotime, we used a simplified approach using only the first coordinate (DC1) of diffusion map. We divided the range of DC1 coordinate into 50 bins of equal length and calculated mi,k, mean log2(TPM + 1), for each ith gene and jth cell combination falling into the kth bin. We filtered out genes for which mi,max ≤ 1, mi,max = max(mi,1, mi,2, …, mi,k). For demonstration, we selected the top 50 variable genes by sorting by σi2, variance for mi,k for ith gene among all bins. The order of genes was determined by the index of Medi (median for mi,k), sorted in ascending order. A standard score or Z score, zi = (mi,kMk)/σi, where Mk = mean(mi,1, mi,2, …, mi,k), was calculated for each gene separately.

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