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Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression

Abstract

Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousands of almost perfectly staged 1-cell stage embryos. As the earliest embryonic events are driven by posttranscriptional regulation, we combined eFACS with second-generation sequencing to profile the embryonic expression of small, noncoding RNAs. We discovered complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including microRNAs and 26G-RNAs, during embryogenesis.

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Figure 1: eFACS yielded tens of thousands of staged 1-cell stage embryos with at least 98% purity.
Figure 2: Dissecting small RNA expression during embryogenesis.
Figure 3: Flow cytometry–based sorting of fixed 1-cell stage embryos revealed miRNA expression dynamics.
Figure 4: Composition of different classes of small RNAs at different developmental stages obtained by deep sequencing.
Figure 5: Small RNA length distribution at different developmental stages.
Figure 6: The 26G-RNAs were expressed from intergenic clusters.

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Acknowledgements

We thank R. Lin (University of Texas) for providing us with the TX189(P(oma-1)oma-1GFP) strain. All other strains used in this project were provided by the Caenorhabditis Genetic Center, which is funded by the US National Center for Research Resources. M.S. acknowledges part-time funding from the Berlin Institute for Medical Systems Biology, funded by Bundesministerium für Bildung und Forschung, and New York University PhD exchange program, and a travel grand from Boehringer Ingelheim Fonds. J.M. thanks the Deutsche forschungsgemeinschaft for a fellowship in the International Research Training Group Genomics and Systems Biology of Molecular Networks (GRK 1360). F.P. and N.R. acknowledge partial funding from US National Human Genome Research Institute (ModEncode U01 HG004276) and US National Institutes of Health (R01HD046236). We thank S. Lebedeva for help with sequencing runs.

Author information

Authors and Affiliations

Authors

Contributions

F.P. and N.R. conceived, designed and supervised the study. M.S. designed and performed the experiments. J.M. designed and performed computational studies with the exception of predicting new miRNAs, which was done by M.R.F.; T.C. contributed to initial eFACS experiments; H.-P.R. helped with flow cytometer settings and runs; W.C. and N.L. contributed to library preparations and sequencing; M.S., J.M., F.P. and N.R. analyzed the data. M.S. and N.R. wrote the paper, and J.M. and F.P. edited it.

Corresponding authors

Correspondence to Fabio Piano or Nikolaus Rajewsky.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–3,5,7–9 (PDF 2957 kb)

Supplementary Table 4

miRNA predictions using miRDeep. The table depicts 19 predicted miRNAs from our datasets including star and precursor sequences. (XLS 46 kb)

Supplementary Table 6

Expression of 21U-RNAs. We retrieved 15,703 21U-RNA sequences from the supplementary material of Batista et al, 2009, and mapped them to the genome. The subset mapping to unique positions in the genome was quantified by reporting weighted mappings of reads that overlap with each 21U-RNA. (XLS 1488 kb)

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Stoeckius, M., Maaskola, J., Colombo, T. et al. Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression. Nat Methods 6, 745–751 (2009). https://doi.org/10.1038/nmeth.1370

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