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Status |
Public on Aug 28, 2024 |
Title |
WT testis, D6-8, scRNAseq |
Sample type |
SRA |
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Source name |
Testis
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Organism |
Drosophila melanogaster |
Characteristics |
tissue: Testis strain: Oregon R genotype: Wild-type developmental stage: Adult age: 6-8 days Sex: Male
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Treatment protocol |
Adult male flies at 3-5, 6-8, 9-11 days of adulthood, and approximately 100 pairs of testes were dissected for each sample preparation. Testes were dissected into Schneider’s medium and then washed twice with cold 0.04% BSA DPBS solution. 500 µL 0.25% Trypsin was added to each tube/sample. Samples were vortexed with 1000rpm for 15-18 minutes at room temperature. The enzymatic reactions were terminated by adding 500 µL DMEM (with >10% FBS). Cell suspensions were filtered through a 40 µm Falcon filter into an Eppendorf tube. Spermatids were filtered out for further enrichment of early germ cells and somatic cyst cells. The efficiency of single cell dissociation methods for fly testes were tested by labeling somatic cells or germline cells with GFP fluorescence. The percentage of each type of GFP-labeled cells were measured, ensuring retrieval of single cell suspensions with reasonable amount of viable germline and somatic cells.
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Growth protocol |
Fly stocks were raised at 25 °C on standard molasses/yeast medium
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Extracted molecule |
polyA RNA |
Extraction protocol |
Approximately 5,000-10,000 cells were washed with 0.04% BSA DPBS for three times and were resuscitated and manually determined to a concentration of 700~1200 cells/ul (viability≥85%). Cells were captured in droplets at a targeted cell recovery of cells. After the reverse transcription step, emulsions were broken and Barcoded-cDNA was purified with Dynabeads, followed by PCR amplification. Amplified cDNA was then used for 10x Genomics chromium single cell 3ʹ gene expression library construction. For gene expression library construction, 50 ng of amplified cDNA was fragmented and end-repaired, double-size selected with SPRIselect beads.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
10x Genomics v3
|
Data processing |
For each scRNA-seq dataset, raw data were aligned to D. melanogaster reference genome dm6 and gene-barcode matrices were generated by CellRanger version 4.0.0 . For scRNA-seq datasets of 6-8 and 9-11 days-old testes, we forced the number of cells (--force-cells=5000). Count data were further processed using the R package Seurat version 3.2.2. High-quality cells that expressed 200–6,000 genes and had mitochondrial content <10% were retained for the downstream analysis. Single-cell RNA-seq datasets of wild-type and mutant testes across different time points were integrated using the standard integration workflow (https://satijalab.org/seurat/v3.0/integration.html). Somatic and germline lineage were found in our data and each cell type further is annotated using curated markers by somatic cells and germline cells subclustering analysis. Assembly: Drosophila melanogaster Release 6 plus ISO1 MT (GCA_000001215.4) Supplementary files format and content: *.barcodes.tsv.gz, *.features.tsv.gz, *.matrix.mtx.gz : filtered raw counts output by CellRanger count Supplementary files format and content: combined_WT_ChinmoST_testes_umi_counts.csv.gz : Merged raw counts table for high-quality cells in all scRNA-seq datasets Supplementary files format and content: combined_WT_ChinmoST_testes_metadata.csv.gz : Metadata for high-quality cells in all scRNA-seq datasets
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Submission date |
Apr 27, 2022 |
Last update date |
Aug 28, 2024 |
Contact name |
Qing Ma |
Organization name |
Shenzhen Institutes of Advanced Technology
|
Street address |
1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, P.R.China
|
City |
Shenzhen |
ZIP/Postal code |
518055 |
Country |
China |
|
|
Platform ID |
GPL25244 |
Series (2) |
GSE201673 |
Chinmo maintains adult stem cell sex identity by directly regulating Doublesex and Insulin signaling [scRNA-seq] |
GSE201716 |
Chinmo maintains adult stem cell sex identity by directly regulating Doublesex and Insulin signaling |
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Relations |
BioSample |
SAMN27914597 |
SRA |
SRX15014602 |