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Nat Protoc. 2013 Jun;8(6):1088-1099. doi: 10.1038/nprot.2013.062. Epub 2013 May 9.

FACS purification of Drosophila larval neuroblasts for next-generation sequencing.

Author information

1
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria.
#
Contributed equally

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.

PMID:
23660757
PMCID:
PMC3930877
DOI:
10.1038/nprot.2013.062
[Indexed for MEDLINE]
Free PMC Article

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