PRJNA375756: The Drosophila Embryo at Single-Cell Transcriptome Resolution
概要: 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.
项目整体设计: Single cell transcriptomics of the early Drosophila embryo were generated using Drop-seq
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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. |
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Dechorionated embryos were staged and stage 6 embryos hand-picked into ice-cold PBS-Triton 0.1%. fixation: 80% methanol/PBS |
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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 |
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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 |
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poly(A)+ RNA |
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PAIRED |
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Forward |
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ILLUMINA |
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Illumina NextSeq 500 |
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Specific |
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加标(Spike-In) |
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The Drosophila embryo at single-cell transcriptome resolution.
Science (New York, N.Y.) . 2017-08-31 [PMID:
28860209]