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| Status |
Public on Aug 31, 2021 |
| Title |
pros-RNAi E(z)-RNAi neuroblast tumors |
| Sample type |
SRA |
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| Source name |
neuroblast tumors induced by RNAi mediated inactivation of prospero (pros) and Enhancer of Zeste (E(z)) using poxn-GAL4
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: adult 5-7 days-old
genotype: poxn-GAL4, uas-dicer2, UAS-GFP, UAS-prospero-RNAi, UAS-E(z)-RNAi,
fly stock used: uas-prosRNAi (Bloomington # 26745), UAS-E(z)-RNAi (VDRC KK107072)
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| Growth protocol |
Fly stocks were raised at 18°C on standard food (8 % cornmeal, 8 % yeast and 1 % agar). Crosses were performed at 29°C and the progeny were maintained at 29°C to maximize RNAi-mediated knockdown efficiency
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| Extracted molecule |
total RNA |
| Extraction protocol |
Adult flies were killed in 70% ethanol and washed once in PBS. VNCs were dissected in PBS, collected in a RNase-free Protein LoBinding tube filled with ice-cold PBS, and incubated in a freshly prepared dissociation solution containing 0,4% bovine serum albumin (BSA), 1 mg/mL collagenase I and papain (Sigma Aldrich) in PBS for 75 min at 29°C with low agitation. Tissues were then disrupted manually by pipetting up and down with a 200 µL tip. Dissociated cells were pelleted for 20 min at 300 g at 4°C to remove the dissociation solution and resuspended in ice-cold PBS + 0,4% BSA. The cell suspension was filtered through a 30 µm mesh Pre-Separation Filter (Miltenyi) to remove debris and transferred in new RNase-free Protein LoBinding tube for FACS sorting. Forty thousand GFP+ tNBs cells were isolated using a FACSAriaII machine (BD) with an 85 μm nozzle, at 45 psi low pressure and according to viability, cell size and GFP intensity. In the next 30 min, sorted-cells were encapsidated using the Chromium Single Cell Controller (10X Genomics) for single-cell RNA-seq.
Single cells were processed using the Single cell 3’ Library, Gel beads and multiplex kit (10X Genomics, Pleasanton) as per the manufacter’s protocol. Cells were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) with the Chromium Single Cell Controller (10X genomics, Pleasanton), where all generated cDNA share a common 10x Barcode. Libraries were generated and sequenced from the cDNA and the10x Barcodes are used to associate individual reads back to the individual partitions. Analysis using molecular indexing information provides an absolute digital measurement of gene expression levels. Sequencing was performed using a NextSeq 500 Illumina device (1sample) containing transcript lengh of 57 bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Single-cell mRNA seq data were analyzed using the 10x Genomics suite Cell Ranger 2.0.1 with default settings for de-multiplexing, aligning reads to the Drosophila genome (10X Genomics pre-built dm6 reference genome) with STAR and counting unique molecular identifiers (UMIs) to build transcriptomic profiles of individual cells. This first level of analysis generates quality metrics (Q30, number of reads by sample…), FASTQ files and filtered genes matrices. Cell Ranger pre-processing retained 5796 cells with a median number of 1806 genes/cell for the control condition (Genovese, Clement, et al., 2019), 2977 cells with a median number of 1959 genes/cell for the E(z)RNAi condition, 2410 cells with a median number of 700 genes/cell for the trxRNAi condition and 3942 cells with a median number of 1190 genes/cells for the E(z)RNAi, trxRNAi condition. Filtered gene matrices generated via Cell Ranger 2.0.1
Genome_build: dm6 reference genome
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| Submission date |
Jun 29, 2021 |
| Last update date |
Sep 02, 2021 |
| Contact name |
Cédric Maurange |
| E-mail(s) |
[email protected]
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| Organization name |
CNRS
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| Department |
Developmental Biology Institute of Marseille (IBDM)
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| Street address |
Parc Scientifique de Luminy
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| City |
Marseille |
| ZIP/Postal code |
13009 |
| Country |
France |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE179154 |
Single-cell RNA-seq of Drosophila neuroblast tumors induced by the knockdown of prospero, E(z) and trx |
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| Relations |
| BioSample |
SAMN19951724 |
| SRA |
SRX11308618 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5410735_EzRNAi_dim30.rds.gz |
69.3 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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