Abstract
RNA-seq or transcriptome analysis of individual cells and small-cell populations is essential for virtually any biomedical field. It is especially critical for developmental, aging, and cancer biology as well as neuroscience where the enormous heterogeneity of cells present a significant methodological and conceptual challenge. Here we present two methods that allow for fast and cost-efficient transcriptome sequencing from ultra-small amounts of tissue or even from individual cells using semiconductor sequencing technology (Ion Torrent, Life Technologies). The first method is a reduced representation sequencing which maximizes capture of RNAs and preserves transcripts’ directionality. The second, a template-switch protocol, is designed for small mammalian neurons. Both protocols, from cell/tissue isolation to final sequence data, take up to 4 days. The efficiency of these protocols has been validated with single hippocampal neurons and various invertebrate tissues including individually identified neurons within a simpler memory-forming circuit of Aplysia californica and early (1-, 2-, 4-, 8-cells) embryonic and developmental stages from basal metazoans.
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Acknowledgements
We would like to thank our collaborators Drs. Thomas Foster, C. Jason Frazier, Scott Harden and Mrs Elena Bobkiva (University of Florida) for the single neuron isolations and help with dissections. We also want to thank Alexander Fodor for help in single-cell library construction. We thank Mr. James Netherton for reading and commenting on the manuscript. We thank Dr. Manfred Lee for his technical advice and guidance with the Ion PGM sequencing process. We also thank Miss Brandi McLaughlin for help with her guidance and updates for semiconductor sequencing technologies and novel chips as well as for providing photos and schematic diagrams for Figs. 1a–c and 5. This work is supported by NIH grants 1R01GM097502, R21RR025699, 5R21DA030118, R01MH097062, McKnight Brain Research Foundation, Florida Biodiversity Institute as well as NSF-0744649, NSF CNS-0821622, and UF Opportunity Fund awards to LLM.
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Kohn, A.B., Moroz, T.P., Barnes, J.P., Netherton, M., Moroz, L.L. (2013). Single-Cell Semiconductor Sequencing. In: Tollefsbol, T. (eds) Biological Aging. Methods in Molecular Biology, vol 1048. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-556-9_18
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DOI: https://doi.org/10.1007/978-1-62703-556-9_18
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