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| Status |
Public on Jan 12, 2023 |
| Title |
single-embryo Library 2 |
| Sample type |
SRA |
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| Source name |
single-embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: single-embryo
developmental stage: early development (less than or equal to 3 hours)
genotype: WT
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| Growth protocol |
Drosophila genetic reference panel (DGRP) 737 line from Bloomington Stock Center (#83729) was kept in incubators at 25 °C with 60% humidity and a 12-hour light-dark cycle. All flies were raised at constant densities on standardized cornmeal food (Bloomington recipe), Fly food M (LabExpress, Michigan, USA), and transferred into cages 1-2 days after eclosion.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single embryos were homogenized in 500 µL TRIzol™ (Invitrogen, USA) by bead-beating with 0.2 g lysing matrix D beads (MP Biomedicals, USA) at 6 m/s for 30 seconds using the FastPrep-24™ instrument (MP Biomedicals, USA). RNA was then isolated following a miniaturized version of the manufacturer's instructions. Briefly, 100 µL chloroform (Sigma-Aldrich, USA) was added, samples mixed by vortex, incubated 2 min at room temperature (RT), and centrifuged for 15 minutes at 12,000 × g at 4 °C to recover RNA-containing aqueous phase in a fresh 1.5 ml microtube. RNA was precipitated by adding 250 µL ice-cold isopropanol (Sigma-Aldrich, USA) and 2 µL GlycoBlue™ (Thermo Fisher, USA), samples mixed by hand, incubated for 10 min at RT, and centrifuged for 10 minutes at 12,000 × g at 4°C. RNA pellets were washed with 1 ml 75% (v/v) ethanol (Pharmco, USA), dried and stored at -80 °C until further use. This same protocol was followed to isolate RNA from fixed embryos using 1 ml TRIzol™ and proportional changes in chloroform and isopropanol.
RNA-seq was carried out following a miniaturized version of the sensitive highly-multiplexed single-cell RNA-seq (CEL-Seq2) protocol (Hashimshony et al. 2016) using the I.DOT liquid handler (CELLINK). Dried RNA was resuspended in 8 µL nuclease-free water (Invitrogen, USA) and 120 nL dispensed into a 384-well plate holding 240 nL of primer-mix including 192 different cell barcodes with unique molecular identifiers (UMI). Subsequent steps and reagents are detailed in Sagar et al., 2018, except that libraries were diluted 1:10 (cDNA:H2O) before an 11-cycle amplification.
Modified CelSeq2 protocol
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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 |
RNA-seq reads were demultiplexed, mapped, UMI-deduplicated, and counted using STAR 2.7.8a (mode STARsolo).
Raw gene counts files were merged into a single file and gene symbols were updated using the 'merge' function in base R and release FB2022_04.
The gene counts were analyzed using the RaceID3/StemID2/FateID workflow (Herman et al. 2018). Embryos with less than 250,000 reads or transcripts with < 3 read counts on < 5 samples were filtered out from the analysis (n=122). Unfertilized eggs (n=117) and embryos older than 3h were removed, leaving a total of 84 embryos in the final pseudo-time analysis. Well IDs were converted to embryo IDs based on the meta data described in the paper.
Assembly: Berkeley Drosophila Genome Project assembly release 6.28
Supplementary files format and content: Tab-delimited files containing the raw gene count files.
Supplementary files format and content: comma-separated file containing the normalized counts used for pseudotime analysis.
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| Submission date |
Sep 24, 2022 |
| Last update date |
Jan 12, 2023 |
| Contact name |
Adelheid Lempradl |
| E-mail(s) |
heidi.lempradl@vai.org
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| Phone |
6162345846
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| Organization name |
Van Andel Institute
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| Department |
Metabolism and Nutritional Programing
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| Street address |
333 Bostwick Ave NE
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| City |
Grand Rapids |
| State/province |
Michigan |
| ZIP/Postal code |
49503 |
| Country |
USA |
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| Platform ID |
GPL25244 |
| Series (1) |
| GSE214118 |
Gene expression profile at single embryo level of 0-3h old Drosophila embryos |
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| Relations |
| BioSample |
SAMN31140307 |
| SRA |
SRX17694628 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6599296_Sample2.STARsolo_raw.counts.txt.gz |
1.2 Mb |
(ftp)(http) |
TXT |
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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