|
| Status |
Public on Sep 19, 2021 |
| Title |
w1118 single-cell RNA-Seq dataset |
| Sample type |
SRA |
| |
|
| Source name |
Whole ovarian tissue
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Ovary
age: 3 days after eclosion
genotype: w1118
treatment: no treatment
|
| Treatment protocol |
Flies were transferred to 29°C incubator for 1 (24h), 3 (72h) and 4 (96h) days depending on the dataset, and were subsequently dissected.
|
| Growth protocol |
100 adult ovaries from 50 female Drosophila melanogaster flies maintained at 25°C, with access to males and yeast supplements for 3 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For bulk-RNA sequencing: Whole ovaries were extracted from 40 females in complete medium (Grace’s Insect Basal Medium supplemented with 15% FBS. The anterior part of the ovaries were carefully removed before stage 10 egg chambers. Samples were transferred to a steril Eppendorf tube and media was aspirated. Samples were flash frozen using liquid nitrogen and stored until day of the library preparation in -80°C. Total RNA was extracted using Trizol. For single-cell RNA sequencing: Whole ovaries were extracted from female flies that were dissected in complete medium (Grace’s Insect Basal Medium supplemented with 15% FBS) and were transferred to a tube containing 300 μL EBSS (no calcium, magnesium, and phenol red), followed by a gentle wash for 2 minutes. The EBSS was then removed and the tissue was dissociated in 100 μL Papain (50 U/mL in EBSS and previously heat activated in 37°C for 15 minutes) for 30 minutes. The suspension was mechanically dissociated every 3 minutes by gentle pipetting up and down. To quench the digestion, 500 μL complete medium was added to dissociated cells. The suspension was then passed through a 40 μL sterile cell strainer and centrifuged for 10 minutes at 700 RCF to remove large eggs with intact eggshell which cannot be dissociated and debris. Supernatant was removed and single cells were re-suspended in 100 μL. Cell viability was assayed using Trypan Blue and estimates of cell concentration were made using a hemocytometer. Cells were then further diluted to an approximate, final concentration of 2,000 cells/μL according to 10X Genomics recommendations.
Bulk sequencing libraries were made using the Ultra II Directional RNA library prep kit for Illumina using the protocol for NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB#E7490). Single-cell libraries were prepared using the Single Cell 3’ Library & Gel Bead Kit v2 and Chip Kit according to the recommended 10X Genomics protocol. Single cell suspension was loaded onto the Chromium Controller (10X Genomics). Library quantification assays and quality check analysis was performed using the 2100 Bioanalyzer instrument (Agilent Technologies). The library samples were then diluted to a 10nM concentration and loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument flow cell for a 100-cycle sequencing run.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Illumina Casava1.7 software used for base-calling
Single-ended fastq files for the whole-tissue RNA-Seq samples were aligned to Drosophila melanogaster Release 6 (Drosophila_melanogaster.BDGP6.28.100.chr_filtered.gtf) reference genome assembly using STAR (v2.7) aligner. featureCounts (Subread package) was run on individual bam files for the generation of count-matrix.
Genome index for single-cell analysis was built using the Drosophila melanogaster Release 6 (Drosophila_melanogaster.BDGP6.28.100.chr_filtered.gtf) reference genome assembly using 10X genomics cellranger (version 3.0.1) mkref pipeline. Sequenced reads were then mapped to this reference genome index. The cellranger count pipeline for alignment, filtering, barcode counting and UMI counting was used to generate the multidimensional feature-barcode matrix for single-cell RNA-Seq datasets.
Genome_build: dm6
Supplementary_files_format_and_content: csv files contain raw counts for each whole-genome RNA Seq sample.
Supplementary_files_format_and_content: 3 files were obtained after running cellranger pipeline (barcodes.tsv, features.tsv and matrix.mtx) on the single-cell RNA-Seq datasets. All these files are readable in R and were processed using the Seurat pipeline which resulted in the individual R object files for each corresponding dataset.
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| |
|
| Submission date |
Aug 20, 2021 |
| Last update date |
Sep 20, 2021 |
| Contact name |
Wu-Min Deng |
| E-mail(s) |
wdeng7@tulane.edu
|
| Organization name |
Tulane University
|
| Department |
School of Medicine, Department of Biochemistry
|
| Street address |
1700 Tulane Avenue
|
| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70112 |
| Country |
USA |
| |
|
| Platform ID |
GPL25244 |
| Series (1) |
| GSE182505 |
Modeling Notch-induced tumor cell survival in the Drosophila ovary identifies cellular and transcriptional response to nuclear NICD accumulation |
|
| Relations |
| BioSample |
SAMN20874989 |
| SRA |
SRX11836916 |
