Pre-filtering, total droplet-encapsulated cell count was 78,551, summing 16,279 FRE, 24,850 FBS, 27,756 REC, and 9666 DNA. Post-filtering by gene and mitochondrial transcript count led to an overall cell count reduction of 1%. 21,117 genes were detected on average across all samples, with 19,173 in FRE, 19,428 in FBS, 19,747 in REC, and 17,416 in DNA. The pre-filtration average unique molecular identifiers (UMI) were 4576 across all samples, with 6,230 in FRE, 4062 in FBS, 3,788 in REC, and 5248 in DNA. Filtration led to an increase in overall average UMI by 1%. Pre-filtered average percent mito-based reads were 2.6% overall, with 1.9% in FRE, 2.4% in FBS, 3.5% in REC, and 2.6% in DNAse. Filtration led to a decrease in average percent mitochondrial reads of 1%. No significant differences were found between treatment conditions, or in comparing pre- vs post-filtration metrics. We focus on comparisons between CSF A, B, and C in our studies since these were the only donors for which the FRE, FBS, and REC conditions were all available. CSF A, B, and C feature count (average genes per cell), UMI count, and percent mitochondrial read distributions are presented in Additional file
1: Fig. S2A-C. The average genes detected per cell in 2041 in FRE, 1102 in FBS and 1273 in REC. There was an overall decrease in the average genes detected per cell in both the cryopreservation conditions, but REC was found to be significant (one way ANOVA, Tukey’s Posthoc test, p = 0.0031). UMI was overall higher in FRE than FBS and REC groups, though this was not significant statistically: F(1.20, 2.401) = 8.86, p = 0.08. There were no differences in the percentage of mitochondrial reads across the three groups (p = 0.94). Features, UMI, and percent mitochondrial reads were very similar between FBS and REC (including samples CSF A-G, see Additional file
1: Fig. S4B), and between FBS and DNA conditions (including samples CSF A, C, D, and G, see Additional file
1: Fig. S5B). See Additional file
1: Table S2 for detailed quality control-based metrics for each donor, condition, and sample replicate. Examination of global heat shock protein genes HSPA1A, HSPA1B, and HSP90AA1 found minimal to no differences in gene expression by cryopreservation method in any of the three donors (Additional file
1: Fig. S2D-F).