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. 2021 Mar 19;12:1758. doi: 10.1038/s41467-021-22021-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Workflow of scRNA-seq experiment. Number of cells collected: 6884 (0 day), 6936 (2 day), 6200 (3 day), and 5587 (4 day). b A t-distributed Stochastic Neighbor Embedding (tSNE) plot for analyzed cells. Cluster 0–3 were germ cells and Cluster 4 was somatic cells. Number of cells selected for analysis: Cluster 0 (6487 cells), Cluster 1 (5286 cells), Cluster 2 (3860 cells), Cluster 3 (2170 cells), and Cluster 4 (285 cells). c Violin plots showing the expression level of representative genes in each cluster. d A bar plot showing the proportion of the different cell clusters at different time points. e A heatmap showing the expression of marker genes and GO functions in each cluster. f Gene expression patterns of indicated genes on tSNE plots and violin plots. Expression levels are calculated by UMI counts. g PCA analysis showing the relationship between single-cell clusters from NRRA-treated culture in vitro (n = 4 clusters) and early mouse spermatogenesis in vivo (n = 6 clusters) based on the transcriptional profiles of commonly expressed genes (n = 7803 genes). h Hierarchical clustering showing the relationship between single-cell clusters from NRRA-treated culture in vitro and early mouse spermatogenesis in vivo. “NR/RA-upregulated” indicates genes that were upregulated by NR or RA. “NRRA-upregulated” indicates that were upregulated by NRRA (not upregulated by NR or RA alone). i Scatter plots comparing marker genes expression profile between in vitro clusters and in vivo clusters. j (Left) Heatmap of sex chromosome genes in Clusters 0–3. (Right) Violin plots showing percentage of X and Y chromosome genes profiles in Clusters 0–3. The median is shown by a red line. k Scatter plots showing cells along the projected pseudo-time in Cluster 0–3 (upper) and in indicated clusters from early mouse spermatogenesis in vivo (lower).