Gene Expression
Nebulas
Gene Expression NebulasSummary: Spermatogenesis has been well studied in rodents and invertebrates, but remains poorly understood in humans. As a step towards illuminating human spermatogenesis, we used single-cell RNA-sequencing (scRNAseq) analysis to analyze neonatal and adult human testes. Clustering analysis of neonatal testes revealed 3 germ subsets, including cells with characteristics of primordial germ cells (PGCs), and more differentiated cells with gene expression profiles similar with adult spermatogonial stem cells (SSCs). We identified markers for these neonatal subsets, including protein markers for the PGC-like (PGCL) subset. Clustering analysis of the adult testis revealed 9 germ and 3 somatic cell subsets. Among the germ cell clusters are 4 undifferentiated spermatogonia (SPG) states, each marked by specific genes. One of the SPG states has characteristics suggesting it is enriched for SSCs. We identified protein markers specific for this state, including cell-surface proteins that we used to enrich for these cells. We mapped the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also defined somatic cell subsets in the human testis and traced their developmental trajectories. Together, our data provides a blueprint for understanding the development of the male germline and supporting somatic cells in humans. The germ cell subset markers we identified are candidates to be used for clinical applications, including SSC therapy for treating infertility.
Overall Design: Single cell sequencing from two neonatal and two adult testicular cells was performed. Cells were either enriched for ITGA6 expression or unfractionated before GEM capture
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Testis tissue was dissociated using two step enzymatic digestion with collagenase-IV and trypsin-EDTA. Single cell suspension was washed and enriched for ITGA6 expressing cells (MACS column) or was direclty used. Cells were diluted for loading on Chromium chip.Chromium Single Cell Kit [v2 chemistry].10x single cell RNA sequencing |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| 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 | Case Detail | Control 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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