Additional file 1 of Postnatal developmental trajectory of sex-biased gene expression in the mouse pituitary gland

Additional file 1: Figure S1. Illustration of 3’UTR-seq method and quality control. A. Overview of the QuantSeq data analysis pipeline. B. Genome browser screenshot of extended 3’UTRs for genes Pou1f1 (left) and Ghrhr (right). Gene name and gene model are shown on the bottom of each panel. Each track represents overlapping signal from 5-6 biological replicates. C. Summary of qPCR vs. 3’UTR-seq comparisons across samples in all samples. Bottom panel: bar plots showing the numbers of genes detected using qPCR (white bars) and 3’UTR seq (red bars). The Spearman correlation coefficients between two experiments are shown for each sample in the dot plot (middle panel) and as well as in a density plot (top panel). Figure S2. Correlation heatmaps for pituitary gland samples. Pearson correlation between samples is calculated based on (A) gene expression and (B) miRNA expression. Hierarchical clustering is performed based on pairwise Pearson correlation coefficients (PCCs). Heatmap color intensity represents PCCs (orange: lower correlation; purple: higher correlation). Sample conditions are shown in top bars. Green shades: different ages, blue: male samples; red: female samples. Figure S3. Characterization of sex-biased mRNAs and miRNAs. A. Gene expression heatmap of sex-biased genes at PD12 and PD22, and genes with significant sex-by-age effect between PD12 and PD22. Each row represents a gene and each column represents a sample. Column annotation bars indicate sample age and sex. Colors represent row-scaled log2 (normalized counts). Whether a gene is female-biased (red) or male-biased (blue) at each corresponding age is summarized by the row annotation bars on the left. Sex chromosome-linked genes are labeled with asterisks. Genes with significant sex-by-age interaction effect between PD12 and PD22 are bolded. B. Expression plots of all sex-biased miRNAs. Log2(normCounts) are plotted for each miRNA across ages. Large filled points represent median expression at each age and unfilled points represent each biological replicate. Red: female samples; blue: male samples. C. novel46 is a mirtron of Cacna1g. The precursor of novel46 is expressed from the last intron of Cacna1g (highlighted in blue). The canonical seed region (nucleotide positions 2-8) in the mature sequence of novel46 is bolded. Figure S4. Summary of gene co-expression modules. Left panel. Module expression profile (log2-transformed normalized counts (log2(normCounts)) scaled per gene across all samples) is plotted for genes within each co-expression module across profiled postnatal ages. Dotted lines: individual gene profiles, solid line: median expression profile of module genes. Number of genes within each module is labeled at the top of the plots. Red: female samples; blue: male samples. Right panel. Expression profile of top five hub genes for each module. log2(normCounts) is plotted across profiled postnatal ages. Large, filled points represent median expression at each age and unfilled points represent each biological replicate. Blue: male samples; red: female samples. Figure S5. Characterization of co-expression gene modules. A. Correlation heatmap based on module eigengene (first PC, obtained using “mod_summary()” function from “CEMitool”). Color scale represents pairwise Pearson correlation coefficients. B. Barplots showing the eigengene for each sample in each module. Samples are grouped by age and colored by sex (blue: male; red: female). C. Barplots showing pathway enrichment results for genes in each module. Each bar represents a pathway. Color represents pathway categories (BP: Biological Process; CC: Cellular Component; MF: Molecular Function). Number of module genes in the pathway is labeled. X axis: -log10(P-value) of each pathway. Figure S6. Co-expression module gene enrichment in single-nuclei RNA-seq data. Uniform Manifold Approximation and Projection (UMAP) dimension reduction representation of adult female and male mouse pituitary gland single-nuclei transcriptome integrated by sex from Ruf-Zamojski et al. 2021 (A) colored by cell type and (B) colored by sample sex. Samples derived from snap-frozen pituitaries were first merged between replicates for each sex (n=3/sex). Merged samples were then integrated between sexes. C. Enrichment heatmap of co-expression module genes within cell types. One-sided Kolmogorov–Smirnov (KS) test was performed to test for enrichment of each co-expression module gene within a given cell type compared to all other cell types. Color gradient represents KS test FDR-adjusted P-value for each gene (dark purple: high enrichment; gray: low enrichment). Only genes with FDR ≤ 0.05 in at least one cell type are plotted and only FDR < 0.05 are shown. Each column represents a cell type as labeled at the bottom of the heatmap. Each row represents a co-expression module gene and the genes are grouped by the co-expression module in which the gene was identified (labeled on the right). Breaks were added in the heatmap between co-expression modules. Colored boxes represent the level of significance based on a one-sided hypergeometric test to determine if a group of module genes with KS test FDR ≤ 0.05 was significantly enriched for a given cell type. D. Estimated cell-type proportions by RNA-seq deconvolution using Proportions in Admixture (WGCNA) changes across profiled ages of pituitary cell types without known sex differences in their proportions. Estimated cell-type proportions are plotted across postnatal ages along the x-axis. Large circles and triangles represent the mean cell-type proportion at each age and small circles and triangles represent each biological replicate. Lighter color, solid line, circle points: female samples; dark color, dotted line, triangle points: male samples. Wilcoxon test was performed to compare cell proportions between both sexes at each age (*P<0.05, **P<0.01). See Figure 5B for estimated proportions of cell types with known sex biases.

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