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. 2019 Mar 19;10:1251. doi: 10.1038/s41467-019-09182-1

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© The Author(s) 2019

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 — Cell-type classification by mapping the first wave of spermatogenesis. a Schematic representation of the major germ cell-types and their corresponding developmental processes. Spermatogonia differentiate undergoing mitotic cell divisions before forming spermatocytes that divide by meiotic division. Spermatids differentiate throughout spermiogenesis to form mature sperm. The timeline in the lower panel indicates at which point during the first wave of spermatogenesis samples were harvested for the generation of scRNA-Seq (X) or bulk RNA-Seq (B) data. b Representative images of seminiferous tubules from PAS-stained tissue cross-sections from animals harvested for scRNA-Seq at different time-points during the first wave of spermatogenesis. Developmental progression is illustrated as tSNE below with juvenile cells (colours corresponding to clusters depicted in Fig. 1e) mapped to cells isolated from adult mice (grey). The approximate timing of the stage and cycle of the tubule is illustrated in the form of a circle (Fig. 1b). Scale bars represent 100 µm; original magnification ×20 or ×40. c The percentage of cells allocated to each cell cluster for each juvenile and adult sample is represented by the size of squares with the colours corresponding to the clusters depicted in Fig. 1e. Cell clusters were labelled according to morphologically-defined cell-types: SG: spermatogonia, eP1/2: early-pachytene spermatocyte (SC) 1/2, mP: mid-pachytene SC, lP1/2: late-pachytene SC 1/2, D: diplotene SC, MI: meiosis I, MII: meiosis II, S1-S11: step 1–11 spermatids, FLC: Fetal Leydig cells, LC: Leydig cells. d tSNE representation of RA-synchronised cells from Chen et al.²¹ mapped to the germ cells from our adult B6 scRNA-Seq data. RA-synchronised cells are labelled throughout the plot as follows: A1: type A1 spermatogonia, In: intermediate spermatogonia, BS: S phase type B spermatogonia, BG2: G2/M phase type B spermatogonia, G1: G1 phase pre-leptotene SC, epL: early-S phase pre-leptotene SC, mpL: mid-S phase pre-leptotene SC, lpL: late-S phase pre-leptotene SC, L: leptotene SC, Z: zygotene SC, eP: early-pachytene SC, mP: mid-pachytene SC, lP: late-pachytene SC, D: diplotene SC, MI: metaphase I, MII: metaphase II, RS1o2: S1–2 spermatids, RS3o4: S3–4 spermatids, RS5o6: S5-6 spermatids, RS7o8: S7-8 spermatids

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