Single cell RNA-seq of pluripotent neoblasts and its early progenies
We isolated X1 neoblasts cells enriched in high piwi-1 expression (Neoblast Population), and profiled ∼7,614 individual cells via scRNA-seq. Unsupervised analyses uncovered 12 distinct classes from 7,088 high-quality cells. We designated these classes Nb1 to Nb12 and ordered them based on high (Nb1) to low (Nb12) piwi-1 expression levels. We further defined groups of genes that best classified the cells parsed into 12 distinct cell clusters to generate a scaled expression heat map of discriminative gene sets for each cluster. Expression of each cluster’s gene signatures was validated using multiplex fluorescence in situ hybridization (FISH) co-stained with piwi-1 and largely confirmed the cell clusters revealed by scRNA-seq.
We also tested sub-lethal irradiation exposure. To profile rare pluripotent stem cells (PSCs) and avoid interference from immediate progenitor cells, we determined a time point after sub-lethal irradiation (7 DPI) with minimal piwi-1+ cells, followed by isolation and single-cell RNA-seq of 1,200 individual cells derived from X1 (Piwi-1 high) and X2 (Piwi-1 low) cell populations (Sub-lethal Irradiated Surviving X1 and X2 Cell Population)
t-SNE plot shows two-dimensional representation of global gene expression relationships among all neoblasts (n = 7,088 after filter). Cluster identity was assigned based on the top 10 marker genes of each cluster (Table S2), followed by inspection of RNA in situ hybridization patterns. Neoblast groups, Nb.
Expression of SMED30009323 (SMED30009323) t-SNE clustered cells
Violin plots show distribution of expression levels for SMED30009323 (SMED30009323) in cells (dots) of each of the 12 neoblast clusters.
Sub-lethal Irradiated Surviving X1 and X2 Cell Population
t-SNE plot of surviving X1 and X2 cells (n = 1,039 after QC filter) after sub-lethal irradiation. Colors indicate unbiased cell classification via graph-based clustering. SL, sub-lethal irradiated cell groups.
Expression of SMED30009323 (SMED30009323) in the t-SNE clustered sub-lethally irradiated X1 and X2 cells.
Violin plots show distribution of expression levels for SMED30009323 (SMED30009323) in cells (dots) of each of the 10 clusters of sub-leathally irradiated X1 and X2 cells.
Embryonic Stages: Y: yolk. S2-S8: Stages 2-8. C4: asexual adult. SX: virgin, sexually mature adult.
For further information about sample preparation and analysis for the single animal RNA-Seq experiment, please refer to the Materials and Methods
