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| Status |
Public on Jun 01, 2023 |
| Title |
wildtype, replicate 1, scRNAseq |
| Sample type |
SRA |
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| Source name |
thorax
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: thorax
Sex: male
genotype: tub-Gal80ts/+ ; dMef2-GAL4/+
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| Treatment protocol |
Male flies were incubated for 4 days at 29°C
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| Growth protocol |
Crosses were kept at 18°C to avoid expression of REPTOR or FoxO during development. Adult males were collected every 48-72 hours and incubated 3-4 days at 18°C before being shifted to 29°C to induce expression of REPTOR or FoxO
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| Extracted molecule |
total RNA |
| Extraction protocol |
After 4 days at at 29°C, around 30 thoraces per genotype were dissected making sure the guts were completely removed, snap frozen and stored at -80°C. Single nuclei extractions were prepared according to Fly Cell Atlas protocol with few modifications (Li et al., 2022). Thoraces were homogenized in 1 ml of homogenization buffer (Li et al., 2022) using 1ml dounce (Wheaton 357538). After filtering the extract with a cell strainer (40 µm) and a 40 µm Flowmi (BelArt, H13680-0040), nuclei were collected via centrifugation for 10 minutes at 1000g at 4 ºC, and then re-suspended in 800 ul PBS/BSA buffer (1xPBS/0.5%BSA with RNase inhibitor). Nuclei solutions were filtered again using 40 µm Flowmi (BelArt, H13680-0040) and stained with DRAQ7™ Dye (Invitrogen, D15106). Single nuclei were sorted using a Sony SH800Z Cell Sorter. Around 400k nuclei were collected, centrifuged for 1000g for 10 minutes at 4 ºC, ressuspended at 800-1000 cells/µl in PBS/BSA buffer, and then processed for snRNA-seq 10x genomics following the manufacturer instructions.
Libraries were generated according to 10X genomics Next GEM Single Cell 3’ Library and Gel Bead Kit v3.1
10x snRNA-seq
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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
wt1
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) using parameter --force-cell=10000.
The counts matrices were then analyzed in Seurat, filtering out cells with nCount <100 and nUMI<100 from downstream analysis
Batch correction was performed using the Harmony algorithm before creating a UMAP and performing clustering analysis.
Assembly: BDGP6.32
Supplementary files format and content: 2023-02-21_normalized_count_Pedro_thorax_10K.csv (normalized count matrix)
Supplementary files format and content: 2023-02-21_metadata_Pedro_thorax_10K_final.csv (cell annotation)
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| Submission date |
Mar 09, 2023 |
| Last update date |
Jul 17, 2023 |
| Contact name |
Yanhui Hu |
| E-mail(s) |
yanhui_hu@hms.harvard.edu
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| Phone |
617-432-6546
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Lab |
Norbert Perrimon
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE227038 |
Transcriptomic analysis of REPTOR or FoxO increased activity in muscle tissue using single nuclear RNA-seq analysis of Drosophila thoraces |
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| Relations |
| BioSample |
SAMN33705814 |
| SRA |
SRX19625141 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7090342_wt1.barcodes.tsv.gz |
49.6 Kb |
(ftp)(http) |
TSV |
| GSM7090342_wt1.features.tsv.gz |
143.3 Kb |
(ftp)(http) |
TSV |
| GSM7090342_wt1.matrix.tsv.gz |
6.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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