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| Status |
Public on Jun 15, 2022 |
| Title |
wing_disc_control_scrna |
| Sample type |
SRA |
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| Source name |
wing imaginal disc
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: wing imaginal disc
developmental stage: thrid instar larvae
condition: control
genotype: w1118; +; rn-Gal4, UASeiger, tub-Gal80ts/TM6B
treatment: none
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| Treatment protocol |
Expression of the oncogenic driver Ras/Scrib was induced by raising the flies at 25°C
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| Growth protocol |
Wild-type flies were raised at 18°C at a density of 50 larvae/vial. Flies with induced Ras/Scrib tumor were raised at 25°C on a yeast-based medium under a 12h-12h day-night light cycle.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Eye-antennal discs or wing discs were dissected and transferred to a tube containing 100 µl ice cold PBS. After centrifugation at 800 g for 5 min, the supernatant was replaced by 50 µl of dispase (3 mg/ml, Sigma-Aldrich_D4818-2mg) and 75 µl collagenase I (100 mg/ml, Invitrogen_17100-017). Discs were dissociated at 25°C in a Thermoshaker (Grant Bio PCMT) for 45 min at 25°C, 500 rpm. The enzymatic reaction was reinforced by pipette mixing every 15 min. Cells were washed with 1 ml ice cold PBS and resuspended in 400 µl PBS supplemented with 0.04% BSA. Cell suspensions were passed through a 10 µM pluriStrainer (ImTec Diagnostics-435001050). Cell viability and concentration were assessed by the LUNA-FL Dual Fluorescence Cell Counter.
Single-cell libraries were generated using the 10x Chromium Single-Cell Instrument and Single Cell 3’ Gene Expression (GEX) kit according to the manufacturer’s protocol. Briefly, single cells from eye-antennal discs or wing discs were suspended in 0.04% BSA-PBS. After generation of nanoliter-scale Gel bead-in-emulsions (GEMs), GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad) programmed at 53°C for 45 min, 85°C for 5 min, and hold at 4°C. After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was then amplified by PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 67°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 14 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready library was cleaned up 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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| Data processing |
Sequencing data were processed with CellRanger (v5.0.1) with default parameters and FlyBase r6.35 reference genome
scRNA data was processed using the NextFlow pipeline VSN (Flerin et al., 2021). VSN processing includes the doublet filtering using scrublet (Wolock et al., 2019) and the correction for batch effects between the runs using Harmony (Korsunsky et al., 2019). We used the following filtering parameters : minimum of 200 detected genes per cell, maximum of 15% signal of mitochondrial origin, minimum of 3 cells expressing any targeted genes.
Assembly: dm6
Supplementary files format and content: CellRanger output for each scRNA sample (control and tumor) comprising GEX features are present as TSV (barcode and features) and matrix (raw counts) format.
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| Submission date |
Jun 02, 2022 |
| Last update date |
Jun 16, 2022 |
| Contact name |
Stein Aerts |
| Organization name |
VIB-KU Leuven
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| Department |
Department of Human Genetics / VIB Center for Brain and Disease Research
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| Lab |
Laboratory of Computational Biology
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| Street address |
Herestraat 49
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| City |
Leuven |
| State/province |
Flemish-Brabant |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL25244 |
| Series (2) |
| GSE205399 |
Shared enhancer gene regulatory networks between wound and oncogenic programs [scRNA] |
| GSE205401 |
Shared enhancer gene regulatory networks between wound and oncogenic programs |
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| Relations |
| BioSample |
SAMN28864619 |
| SRA |
SRX15594854 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6211398_REW_2e692e_10x_rotund_eiger_wounding_wing_ctrl_S2.barcodes.tsv.gz |
2.2 Mb |
(ftp)(http) |
TSV |
| GSM6211398_REW_2e692e_10x_rotund_eiger_wounding_wing_ctrl_S2.features.tsv.gz |
125.6 Kb |
(ftp)(http) |
TSV |
| GSM6211398_REW_2e692e_10x_rotund_eiger_wounding_wing_ctrl_S2.matrix.mtx.gz |
87.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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