Fig. 4

Long-term storage of fixed mouse lung samples. a Mapping analysis of sequencing reads within a genomic region. b Comparative analysis of the number of detected genes (top) and UMIs (bottom) across various sequencing depths. c Cumulative gene counts analyzed using randomly sampled cells. d Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) representation of gene expression profile variances of fixed, cryopreserved, and fixed/cryopreserved samples. e Linear regression model comparing average gene expression levels of expressed genes across protocols used. The coefficient of determination (R²) computed with Pearson correlation is indicated. f Hierarchical clustering of coefficient of determination (R²) obtained for all pair comparisons across protocol for biological hallmarks, including apoptosis, hypoxia, reactive oxygen species (ROS), cell cycle G2/M checkpoint, unfolded protein response (UPR), and inflammatory response genes. g UMAP visualization of 24,291 fixed, cryo, and fixed/cryo mouse lung cells, colored by 20 cell populations. h Comparison of cell population proportions between fixed (n = 10,256), cryopreserved (n = 8609), and fixed/cryopreserved cells (n = 5426). The top figure shows the difference in cell population proportions between fixed, cryo, and fixed/cryo samples, and the bottom figure shows the results of the compositional cell analysis using the Bayesian model scCODA. Credible changes and Log2FC are indicated. i Differential gene expression analysis across conditions: fixed vs cryo (top-left), fixed/cryo vs cryo (top-right), and fixed/cryo vs fixed (bottom). The top differentially expressed genes (DEGs) with significantly adjusted p-values (FDR) < 0.05, upregulated (red), and downregulated (blue) with Log2FC > |1| are indicated