Additional file 1 of Unique Sertoli cell adaptations support enhanced spermatogenesis in chickens

Additional file 1: Fig. S1. Quality control and information of scRNA-seq. A Quality of scRNA-seq of 13 broiler testicular samples, including nFeature_RNA, nCount_RNA and percent.mt. B Information summary of 13 broiler testicular samples sequenced in this study. C A UMAP plot showing the annotated testicular cell types. Each dot represents a single testicular cell and is colored based on the five different ages of broilers. D Thirteen UMAP plots showing the annotated testicular cell types of each broiler sample sequenced in this study with color based on the cell type. E Immunofluorescence staining for SOX9, ACTA2, VWF, CLEC3B, DDX4, TOP2A, TPPP2 (red) and DAPI (blue) in testes. Scale bars = 100 μm. Fig. S2. Marker expression and percentage of each cluster of five different ages. A Marker expression patterns of each cluster on UMAP plots. A gradient of blue and gray represents high or low marker expression levels. B Bar plot showing the percentage of each cluster of five groups (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks). Fig. S3. Dynamic transcriptional characteristics of germ cell development in chicken testes. A Bar plot showing the number of testicular germ cells and percentage of each cluster from five groups (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks). The dashed line represents spermatocytes, while the solid line represents spermatids. B Heatmap showing the markers of each cell cluster of germ cells by “DoHeatmap” function. All markers are calculated by “FindAllMarker” function. The number on the left shows the number of differentially expressed genes and is colored based on its cell type. C UMAP plots showing the annotated cell types of spermatocytes and spermatids. Dashed lines and arrows represent their developmental trajectory. D Heatmaps showing the representative markers of each cell cluster of spermatocytes and spermatids. A gradient of red and blue represents high or low marker expression levels. E Expression patterns of representative dynamic genes as predicted by pseudotime during spermatogenesis. Germ cells are colored based on the cluster colors and ordered according to the pseudotime trajectory. Pseudotime (SSCs to ES2) is indicated below each gene plot column. Fig. S4. Potential markers and molecular characteristics of spermatogonia, spermatocytes, and spermatids in chickens. A–C Heatmap showing the markers of each cell cluster of spermatogonia (A), spermatocytes (B), and spermatids (C). The number on the left shows the number of differentially expressed genes, and the right side shows top 5 markers. D–F Charts showing 5 representative GO terms enriched in the marker genes of each cluster of spermatogonia (D), spermatocytes (E), and spermatids (F) with −log10(P-value) of each GO term. Fig. S5. Pseudotime analysis of spermatogonia, spermatocytes, and spermatids. A–C Pseudotime trajectory of spermatogonia (A), spermatocytes (B), and spermatids (C) revealed cellular states based on the pseudotime. Each dot represents a single germ cell and is colored based on its cell subtype. D–F Heatmap showing the clusters of top 1,000 genes in pseudotime from spermatogonia (D), spermatocytes (E), and spermatids (F) of broiler chickens. A gradient of red and blue represents high or low gene expression levels based on the expression color code. The number on the left side shows the number of differentially expressed genes, and the right side shows the representative genes and GO terms. Fig. S6. Developmental analysis of Leydig cells and Sertoli cells. A Heatmap showing the markers of immature and mature Sertoli cells. The number on the left shows the number of differentially expressed genes, and the right side shows top 5 marker genes. B Bar chart showing 5 representative GO terms enriched in the marker genes of immature and mature Sertoli cells with −log10 (adjusted P-value) of each GO term. C Heatmap showing the markers of precursor and Leydig cells. The number on the left shows the number of differentially expressed genes, and the right side shows top 5 marker genes. D Bar chart showing 5 representative GO terms enriched in the marker genes of precursor and Leydig cells with −log10 (adjusted P-value) of each GO term