Fig. 2

Comparison of cold and warm tissue dissociation protocols. a Bulk RNA-seq profiles of dissociated kidneys. GeTMM-normalized counts [29] were averaged across three biological replicates and log2-transformed after adding a pseudo count of 1. DEGs with FDR < 0.05 and logFC threshold of 2 (edgeR exact test [27]) are shown as red and blue dots; protein-coding genes with logFC > 4 are labelled. b Number of differentially expressed genes (DEGs) between cold- and warm-dissociated scRNA-seq libraries. Calculated for each cell type separately using Wilcoxon test in Seurat [30] with thresholds of logFC = 0.5, minimum detection rate 0.5, FDR < 0.05. Numbers on the right side of the plot indicate cell population size. c Stress score – an expression score for a set of 17 stress-response-related genes (Fosb, Fos, Jun, Junb, Jund, Atf3, Egr1, Hspa1a, Hspa1b, Hsp90ab1, Hspa8, Hspb1, Ier3, Ier2, Btg1, Btg2, Dusp1). Calculated as average gene expression level of these genes subtracted by averaged expression of randomly selected control genes and then averaged for cell types. Significance was calculated in a Monte-Carlo procedure with 1000 randomly selected gene sets of the same size, asterisks denote p value < 0.01. d Expression and detection rates of differentially expressed genes commonly induced in warm-dissociated samples (differentially expressed in at least four cell types). e Cell type composition of freshly profiled scRNA-seq libraries. Three biological replicates are shown per condition. Asterisks denote two-sided chi-square test p value < 0.001. In b–d, podocytes and transitional cells were excluded due to low cell numbers. aLOH: ascending loop of Henle; CD_IC: intercalated cells of collecting duct; CD_PC: principal cells of collecting duct; CNT: connecting tubule; DCT: distal convoluted tubule; PT: proximal tubule