Supplementary MaterialsS1 Fig: Summary of scRNA-seq and transcriptional maps of mouse and NM-R spleens. Gene-by-cell expression-level heatmap of the four-mouse spleen scRNA-seq data, in which selected marker genes are listed to the left and cells are faceted by their converged cluster assignment. (G) Stacked bar chart showing the proportion (%) of cells from each of the four mouse duplicate spleen samples assigned to each of the converged clusters (see S1 Table for underlying data). Samples are color-coded and duplicates are shade-coded. (H) Schematic view showing the workflow in which single-cell suspensions are derived from four (two men [M], two females [F]) NM-R spleens. (I) Club chart showing the amount of cells sequenced in duplicate from each one of the four NM-R spleens (discover S43 Desk for root data). (J) Violin story showing the amount of genes sequenced per cell from each one of the four NM-R spleens (discover S2 Desk for root data). (K) Violin story showing the amount of UMIs sequenced per cell from each one of the four NM-R spleens (discover S2 Desk for root data). (L) UMAP projection from the four NM-R spleen scRNA-seq data, that each true stage is a cell color-coded by its converged-cluster project and annotated cell type. (M) Gene-by-cell expression-level heatmap from the four NM-R spleen scRNA-seq data, where chosen marker genes are detailed left and cells are faceted by their converged cluster project. (N) Stacked club chart displaying the percentage (%) of cells from each one of the four NM-R duplicate spleen examples assigned to each one of the converged clusters (discover S2 Desk for root data). Examples are color-coded and duplicates are shade-coded. DC, dendritic cell; MZ, marginal area; NK, organic killer; NKT, organic killer T; NM-R, nude mole-rat; RP, reddish colored pulp; scRNA-seq, single-cell RNA-sequencing; UMAP, even manifold projection and approximation; UMI, exclusive molecular identifier.(TIF) pbio.3000528.s001.tif (11M) GUID:?9717080B-4E1F-4804-A0CA-EEEC2A3EC2D4 S2 Fig: Distinctions in splenic microarchitecture and gene expression as measured by ISH recapitulate the differences seen in scRNA-seq data. (A) Consultant Rabbit Polyclonal to RED pictures of HE-stained consecutive parts of mouse (higher -panel) and NM-R (lower -panel) spleens proven at 5 (still left sections) and 20 magnifications (best panels), scale club = 100 m. Mouse and NM-R spleens present main distinctions in splenic microanatomy, using the NM-R developing a relatively reduced white-pulp area and a more substantial red-pulp area with a lot more fibromuscular trabeculae that hook up to the capsule and offer structural support and contractility towards the spleen. Inside the white pulp, the marginal area and follicles from the NM-R (which comprise B cells) are easily identifiable. In comparison, the PALS (which comprises the T cellCrich area in other types) is much less prominent in the NM-R than in the mouse. (B) UMAP projections from the mouse (higher panels) and NM-R (lower panels) spleen scRNA-seq data color-coded by LY2801653 (Merestinib) the expression levels of myeloid and lymphoid lineage marker genes: (myeloid marker), (B cell marker, lymphoid), and (T cell marker, lymphoid). (C) Representative consecutive sections of mouse (upper panel) and NM-R LY2801653 (Merestinib) (lower panel) spleens showing results from ISH staining for and the bacterial-specific gene were included as positive and negative controls, respectively (Materials and methods). (D) Quantification of the ISH staining of in mouse (gray bars) and NM-R (pink bars). Bars symbolize the proportion of positive staining cells (= 4 biological replicates) and error bars symbolize the uncertainty associated with the quantity of puncta used as a cutoff for defining a cell as positive for any marker gene (Materials and methods and S5 Table for underlying data). Asterisks mark statistically significant (adjusted 0.05) differences in cell type proportions. Consistent with our findings from your scRNA-seq data, ISH showed a significantly higher level of in the NM-R compared with the mouse, with staining restricted to the red-pulp regions in both species. However, the NM-R has significantly lower large quantity of and is similar between the two species, which is in line with our scRNA-seq data. Despite their comparable abundance, the distribution of positive staining cells is usually markedly different between the two species, with expression in the mouse limited LY2801653 (Merestinib) to the PALS, while that of the NM-R is usually stochastically distributed throughout both the reddish- and white-pulp regions, with only minimal aggregation in the PALS. Thus, direct examination of splenic tissue from mouse and NM-R highlights major differences in microanatomy between the two species and supports our results from the scRNA-seq data with respect to the proportions of the major immune cell lineages in the two species. APC, antigen-presenting cell; DC, dendritic cell; HE, hematoxylin.