Additional file 1 of Single-cell transcriptomic analysis of eutopic endometrium and ectopic lesions of adenomyosis

Additional file 1: Figure S1. HE staining and QC. (A) Samples from the AM_CTRL, AM_EM and AM_EC groups were HE stained, and the black arrow shows the gland invading the muscular layer. (B) X axis represents the number of UMI in each cell, and y-axis represents the number of genes in each cell. The distribution model is fitted according to the linear relationship. Yellow dots indicate cells that deviate from the threshold and will be removed in subsequent analysis. (C) The doublet score increases gradually from light color to dark blue (left figure), the red dots represent doubles predicted by Scrublet (right figure). The proportion of mitochondrial genes (D), the number of genes expressed (E), and the number of UMIs (F) in each cell before and after QC are shown in the violin plots. (G) The mean proportion of mitochondrial genes, mean number of genes expressed, mean number of UMIs in each cell and cell number of the three sample groups before and after QC are shown. HE, Hematoxylin-Eosin; QC, Quality Control. Figure S2. Cell type identification and heatmap of gene expression in clusters. (A) Seventeen clusters were displayed in the AM_CTRL, EAM_EM and AM_EC groups. (B) Heatmap showing the expression levels of specific markers in each cluster. (C) The cell number and percentage corresponding to each cell type were counted. Complementary representative markers of different cell types (D) and corresponding violin plots (E) are shown. Figure S3 Colocalization of epithelial cell and endothelial cell markers in cluster 1. (A, B) Complementary epithelial cell markers (CDH1 and KRT7), endothelial cell markers (VWF and CDH5) and colocalization of the two cell type markers are displayed in the t-SNE map. (C) Confirmatory colocalization of EPCAM and PECAM1 was conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, EPCAM (red), PECAM1 (green) and nuclei (blue) were stained. The white arrows show the colocalized cells containing EPCAM and PECAM1, scale bars = 20 μm, *P < 0.05. GO (D) and KEGG (E) analyses of upregulated genes in cluster 1 compared with all other clusters (cluster 2 to cluster 17). Representative GO (F) and KEGG (G) terms of upregulated genes in cluster 1 compared with the epithelial cell population (cluster 7 and cluster 17) are displayed. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure S4 Gene expression changes and functional analyses of the AM_CTRL, AM_EM and AM_EC groups. Representative GO (A) and KEGG (C) terms of upregulated DEGs in the AM_EC group compared with the AM_EM group are shown. Representative GO (B) and KEGG (D) terms of upregulated genes in the AM_EM group compared with those in the AM_CTRL group are shown. (E) Venn diagram showing the common change genes between AM_EM versus AM_CTRL and AM_EC versus AM_EM. (F) Enriched GO terms of common changed genes in (E) are shown. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure S5. Identification of subclusters in epithelial cell populations. (A) The t-SNE map shows 7 subclusters in the epithelial cell group. Histograms show the proportion of subcluster 1 in AM_EM and AM_EC (B), and the proportion of subcluster 4 is shown in (C). (D) The heatmap displays the expression levels of specific markers in each subcluster. Representative GO (E) and KEGG (F) terms of the top 500 genes expressed in subcluster 1 are indicated. The corresponding GO (G) and KEGG (H) analyses of subcluster 4 are also presented. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. Figure S6. Change in marker distribution on the pseudotime trajectory and verification of VM formation. (A) Distribution of groups on the pseudotime trajectory. (B) Cell type, including epithelial cell, cluster 1 and endothelial cell, distributed on the pseudotime trajectory. (C, D) Epithelial cell markers (EPCAM, CDH1 and KRT7) and endothelial cell markers (PECAM1, VWF and CDH5) distributed on the pseudotime trajectory. (E) Confirmatory VM channel formations were conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, VM channels were positive for PAS staining but negative for CD34 (black arrows); red arrows represent channels positive for CD34; scale bars = 50 μm. (F) Histogram indicates the number of VM channels in different groups; ns represents not significant, *P < 0.05. VM, Vasculogenic Mimicry. PAS, Periodic Acid-Schiff.