Gene Expression
Nebulas
Gene Expression NebulasSummary: The germ cells are vital for reproduction and heredity. However, the mechanisms for female germ cell development in primates, especially in late embryonic stage, has remained elusive. Here, we performed single-cell RNA sequencing of 12471 cells from fetal ovaries. We identified five cell types (germ cell, granulosa cell, theca cell, endothelial cell, macrophage cell), and explored the interactions between germ cells and niche cells. Interestingly, we demonstrated that two waves of oogenesis occur during fetal ovary development and ZGLP1 could activate oogenic program and is essential for meiosis initiation. Furthermore, late formed double strand breaks (DSBs) mediated by PRDM9 may lead to the apoptosis of germ cells during cyst breakdown process. Moreover, our study identified the origin of theca cells that may derive from Leydig cell like cells in fetal ovaries. Overall, our work provides new insights into the molecular and cellular basis of fetal ovary development at single-cell resolution.
Overall Design: Single cell RNA sequencing on fetal ovary cells was performed from Macaca fascicularis.
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| Growth Protocol: | - |
| Treatment Protocol: | -; The ovaries samples for scRNA-seq were from two fetal female Macaca fascicularis (embryonic day 84 and 116). For each single cell sequencing experiment, two ovaries were washed twice in 1× PBS, and subjected to a standard digestion procedure through Tumor Dissociation Kit, human (Miltenyi Biotec # 130-095-929). First, the ovaries were cut into small pieces of 2–4 mm, and transferred into the gentleMACS C Tube containing the enzyme mix (4.7 mL DMEM, 200 µL Enzyme H, 100 µL Enzyme R, and 25 µL Enzyme A). Then, the C Tube was attached onto the sleeve of the gentleMACS dissociator. After termination of the program, C Tube was detached from the gentleMACS Dissociator. The sample was incubated for 30 minutes at 37 °C under continuous rotation using the MACSmix Tube Rotator. Next, C Tube was attached onto the sleeve of the gentleMACS dissociator and then perform a short centrifugation step to collect the sample material at the bottom of the tube. Resuspend sample and apply the cell suspension to a MACS SmartStrainer (70 µm) placed on a 50 mL tube and wash cell MACS SmartStrainer (70 µm) with 20 mL DMEM. Finally, centrifuge cell suspension at 300×g for 7 minutes, aspirate supernatant completely and resuspend cells as required for further applications. |
| Extract Protocol: | Cell instrument to generate a single cell Gel bead in Emulsion(GEM). Single cell RNA libraries were prepared for sequencing according to standard Illumina protocols accompanying the Single Cell 3’ Reagent Kits v3 (10X Genomics)","Cell instrument to generate a single cell Gel bead in Emulsion(GEM). Single cell RNA libraries were prepared for sequencing according to standard Illumina protocols accompanying the Single Cell 3’ Reagent Kits v3 (10X Genomics); RNAs were prepared for sequencing |
| Library Construction Protocol: | Then, count matrix was imported into the R package Seurat and quality control was performed to remove outlier cells and genes. Cells with 200-3000 detected genes were retained. Genes were retained in the data if they were expressed in ≥ 3 cells. After applying these quality control criteria, 11742 cells and 19204 genes remained for downstream analyses. Additional normalization was performed in Seurat on the filtered matrix to obtain the normalized count. Highly variable genes across single cells were identified and principal component analysis (PCA) was performed to reduce the dimensionality on the top 18 principal components. Then, cells were clustered at a resolution of 0.6 and visualized in 2-dimension using Uniform Manifold Approximation and Projection (UMAP)." "Then, count matrix was imported into the R package Seurat and quality control was performed to remove outlier cells and genes. Cells with 200-3000 detected genes were retained. Genes were retained in the data if they were expressed in ≥ 3 cells. After applying these quality control criteria, 11742 cells and 19204 genes remained for downstream analyses. Additional normalization was performed in Seurat on the filtered matrix to obtain the normalized count. Highly variable genes across single cells were identified and principal component analysis (PCA) was performed to reduce the dimensionality on the top 18 principal components. Then, cells were clustered at a resolution of 0.6 and visualized in 2-dimension using Uniform Manifold Approximation and Projection (UMAP).; Cell instrument to generate a single cell Gel bead in Emulsion(GEM). Single cell RNA libraries were prepared for sequencing according to standard Illumina protocols accompanying the Single Cell 3’ Reagent Kits v3 (10X Genomics) |
| Molecule Type: | Poly(A)+ RNA; polyA(+) RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | Specific; - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Condition Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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