Fig. 1

FixNCut protocol in human peripheral blood mononuclear cells (PBMCs). 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 fresh and fixed samples. e, f Linear regression model comparing average gene expression levels of expressed genes (e) and main biological hallmarks, including apoptosis, hypoxia, reactive oxygen species (ROS), cell cycle G2/M checkpoint, unfolded protein response (UPR), and inflammatory response genes (f). The coefficient of determination (R²) computed with Pearson correlation and the corresponding p-value are indicated. g UMAP visualization of 17,483 fresh and fixed PBMCs, colored by 19 cell populations. h Comparison of cell population proportions between fresh (n = 9754) and fixed (n = 7729) PBMCs with the Bayesian model scCODA. Asterisks (*) indicate credible changes. i Differential gene expression analysis between fresh and fixed samples. The top differentially expressed genes (DEGs) with significant adjusted p-values (FDR) < 0.05, upregulated (red), and downregulated (blue) with Log2FC > |1| are indicated. j Violin plot of ex-vivo blood handling gene signature score [19] for fresh and fixed human PBMCs. Statistical analysis between fixed and fresh cells was performed using the Wilcoxon signed-rank test. k Dotplot showing the average expression of sampling-time DEGs for Fresh (y-axis) for all 19 cell types (x-axis) split by protocol. The dot size reflects the percentage of cells in a cluster expressing each gene, and the color represents the average expression level