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FACS purification of Drosophila larval neuroblasts for next-generation sequencing

Nature Protocols volume 8, pages 1088–1099 (2013)Cite this article

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Abstract

Elegant tools are available for the genetic analysis of neural stem cell lineages in Drosophila, but a methodology for purifying stem cells and their differentiated progeny for transcriptome analysis is currently missing. Previous attempts to overcome this problem either involved using RNA isolated from whole larval brain tissue or co-transcriptional in vivo mRNA tagging. As both methods have limited cell type specificity, we developed a protocol for the isolation of Drosophila neural stem cells (neuroblasts, NBs) and their differentiated sibling cells by FACS. We dissected larval brains from fly strains expressing GFP under the control of a NB lineage–specific GAL4 line. Upon dissociation, we made use of differences in GFP intensity and cell size to separate NBs and neurons. The resulting cell populations are over 98% pure and can readily be used for live imaging or gene expression analysis. Our method is optimized for neural stem cells, but it can also be applied to other Drosophila cell types. Primary cell suspensions and sorted cell populations can be obtained within 1 d; material for deep-sequencing library preparation can be obtained within 4 d.

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Figure 1: Schematic overview of the FACS purification protocol for transcriptome analysis of larval neuroblasts (NBs) and neurons, and dissection of larval brains.
Figure 2: Pure populations of NBs and neurons can be separated by FACS.
Figure 3: Representative flow cytometry data to illustrate the gating strategy for FACS purification of larval NBs and neurons.

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Acknowledgements

We thank G. Stengl, T. Lendl and N. Corsini for FACS, P. Pasierbek for imaging support, T.R. Burkard for bioinformatics analyses, and all members of the Knoblich group for discussion and suggestions. We are grateful to the Campus Science Support Facilities Next Generation Sequencing Unit for performing library preparation and next-generation sequencing. C.B. is supported by an EMBO Long Term Fellowship. Work in J.A.K.'s laboratory is supported by the Austrian Academy of Sciences, the Austrian Science Fund, the EU FP7 network EuroSystems and an advanced grant from the European Research Council.

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  1. Christian Berger & Ryan Conder

    Present address: Present addresses: Institute of Genetics, University of Mainz, Mainz, Germany (C.B.); STEMCELL Technologies, Vancouver, British Columbia, Canada (R.C.).,

  2. Heike Harzer and Christian Berger: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna, Austria

    Heike Harzer, Christian Berger, Ryan Conder, Gerald Schmauss & Juergen A Knoblich

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  1. Heike Harzer
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  2. Christian Berger
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Contributions

C.B., H.H. and J.A.K. designed the study. H.H., C.B. and R.C. conducted the experiments. G.S. assisted in the development of the FACS protocol. H.H., C.B. and J.A.K. wrote the manuscript.

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Correspondence to Juergen A Knoblich.

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Harzer, H., Berger, C., Conder, R. et al. FACS purification of Drosophila larval neuroblasts for next-generation sequencing. Nat Protoc 8, 1088–1099 (2013). https://doi.org/10.1038/nprot.2013.062

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