Gene Expression
Nebulas
Gene Expression NebulasSummary: By the onset of morphogenesis, Drosophila embryos consist of about 6000 cells that express distinct gene combinations. Here, we used single-cell sequencing of precisely staged embryos and devised DistMap, a computational mapping strategy to reconstruct the embryo and to predict spatial gene expression approaching single-cell resolution. We produce a virtual embryo with about 8000 expressed genes per cell. Our interactive “Drosophila-Virtual-Expression-eXplorer” (DVEX) database generates three-dimensional virtual in situ hybridizations and computes gene expression gradients. We used DVEX to uncover patterned expression of transcription factors and long noncoding RNAs, as well as signaling pathway components. Spatial regulation of Hippo signaling during early embryogenesis suggests a mechanism for establishing asynchronous cell proliferation. Our approach is suitable to generate transcriptomic blueprints for other complex tissues.
Overall Design: Single cell transcriptomics of the early Drosophila embryo were generated using Drop-seq
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| Growth Protocol: | Egg lays occurred in 1 hour intervals; embryos were aged for 2:30 hours (D. melanogaster) or 3:30 hours (D. virilis) at room temperature. |
| Treatment Protocol: | Dechorionated embryos were staged and stage 6 embryos hand-picked into ice-cold PBS-Triton 0.1%. fixation: 80% methanol/PBS |
| Extract Protocol: | Embryos were dissociated first in a Dounce homogenizer and then further using a syringe with 22G x 2" needle, followed by filtering through 20 um cell strainer and fixation in 80% methanol/PBS |
| Library Construction Protocol: | Captured transcriptomes were reverse transcribed while bound on barcoded oligo dT beads; PCR handles added by template switching; cDNA PCR-amplified (4 + 9 cycles); 600 pg cDNA library fragmented and amplified with Nextera XT v2 DNA sample preparation kit using custom primers for 3'-targeted amplification (Macosko et al. 2015 Cell 161)Drop-seq single-cell transcriptome profiling according to Macosko et al. 2015 Cell 161 |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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