every pair of clusters can be distinguished by at least 5 differentially expressed genes (2-fold change, adjusted Pvalue < 0.01) whose sum of –log10(adjusted Pvalue) is greater than 40 (each gene’s contribution capped at 20). We produced cluster heatmaps at each age group by selecting the top 20 differentially expressed genes in both directions between every pair of clusters. Top markers (Figure 4, 5) were selected manually from the top differentially expressed genes between clusters such that every pair of clusters differ by at least one marker. For comparison of gene expression between clusters, we used the R “limma” package to calculate raw and adjusted P values based on average expression of a given gene within each cluster (log2(TPM+1)).
