Additional file 1 of Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

Additional file 1 : Supplemental Figure 1. Quality control (QC) metrics of single cell data between cryopreserved (Cryostor CS10®, pink) and fresh (RPMI, blue) samples before filtering techniques are applied. QC metrics, including A) the number of unique genes, B) the number of total molecules, and C) the percentage of reads that map to the mitochondrial genome, all demonstrate equivalence between preservation methods before filtering and normalization with D) patient representation between clusters from three patients demonstrated (P1 – SC222, SC223, P2 – SC267, SC268 and P3 – SC272, SC273; Cryostor and RPMI samples respectively). Supplemental Figure 2. Heat map of single cell data clustering of combined cryopreserved (Cryostor CS10®) and fresh (RPMI) samples after filtering techniques are applied. Graph shows the top 5 expressed genes per the 9 identified cell groupings in the dataset. Supplemental Figure 3. Without filtering methods, samples maintain even disbursement with clustering via t-Distributed stochastic neighbor embedding (t-SNE). A) Three patients overlap well with cellular transcriptomic expression across the cell clusters. B) Cryostor® (frozen) and RPMI (fresh) preservation methods show even dispersion across cell clusters. C) Individual patient with paired frozen and fresh specimens demonstrate even dispersion. These t-SNE plots represent 15,910 skin cells, derived from 3 patients with LS (3 fresh and 3 cryopreserved samples with 9245 and 6665 cells respectively). Supplemental Figure 4. Correlation of average genetic expression for major cell groups shows high correlation between sample types. Fresh and cryopreserved samples correlated significantly within cell groups including keratinocytes, T/NK cells, DC/macrophages, fibroblasts, and pericytes even without filtering and normalization. Each point on the correlation plots display the average UMI counts for each gene across all cells for each major cell group. Supplemental Figure 5. Gene expression profiling of known keratinocyte sub clusters from He et al. 2020 were used to define cell clusters. Subclustering of keratinocytes revealed 12 distinct groups of cells within this group which were further identified using defined gene signatures. Gene signatures are presented via feature plot. Supplemental Figure 6. t-Distributed stochastic neighbor embedding plot for 4252 keratinocytes, derived from 3 patients with LS (3 fresh and 3 cryopreserved samples with 3254 and 998 cells respectively). After normalization, tSNE plots show relatively even dispersion of different processing type in each cluster given the much larger overall number of fresh keratinocytes compared to cryopreserved. Bottom separated by patient. Supplemental Table 1. Transcriptomic expression of genes within cell types were similar between preservation methods in frozen media (Cryostor® CS10) compared to fresh media (RPMI). Supplemental Table 2. Wilcoxon ranked statistical testing between Cryostor® and fresh cell numbers demonstrated no significant difference between preservation method. Supplemental Table 3. Differentially expressed genes between Cryostor® and fresh skin samples.