Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

Abstract

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.

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Figure 1: The TIVA tag is a multifunctional, caged mRNA-capture molecule.
Figure 2: Validation of the TIVA tag in solution.
Figure 3: Validation of uptake and uncaging of TIVA tag in live cells.
Figure 4: TIVA tag capture of mRNA from single neurons in mouse hippocampal slices.
Figure 5: TIVA tag capture of mRNA from cells in human live brain tissue specimen obtained from biopsy of the right frontal cortex from a subject undergoing surgery for communicating hydrocephalus.
Figure 6: Bimodal transcripts in single hippocampal neurons in tissue and in culture.

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Acknowledgements

We thank J. Cheung-Lau for assistance with in vitro FRET measurements. Funding was provided by the PhRMA foundation to D.L., US National Institutes of Health (NIH) R01 GM083030 to I.J.D., McKnight Foundation Technology Innovations Award to I.J.D. and J.E., U01MH098953 to J.K. and J.E. and NIH DP004117 to J.E. This project is funded, in part, by the Penn Genome Frontiers Institute under a grant with the Pennsylvania Department of Health, which disclaims responsibility for any analyses, interpretations or conclusions.

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All authors contributed to the writing of the manuscript. D.L. performed dispersed-cell TIVA experiments and some computational analysis; B.K.R. contributed TIVA-tag characterization and supplied TIVA tag; J.L. perfomed slice experiments and the bulk of TIVA uncaging; H.D. performed the bulk of the computational analysis; T.K.K. performed TIVA-mediated RNA amplifications; S.F. contributed computational analysis; C.F. contributed some control samples; J.M.S. contributed TIVA-mediated RNA amplifications; J.A.W., M.S.G. and A.V.U. organized human tissue use; S.B.Y. and J.C.G. contributed TIVA tag; P.T.B. contributed TIVA-mediated RNA amplifications; J.K. directed the computational analysis; J.Y.S. contributed the TIVA uncaging parameters and oversaw the biophotonics; I.J.D. designed experiments and contributed oversight of TIVA-tag synthesis; J.E. designed experiments and contributed oversight of the biological experiments.

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Correspondence to James Eberwine.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1, 2, 4 and 5 (PDF 4202 kb)

Supplementary Table 3

Environment specific expressed genes in single neurons from culture vs. tissue (XLSX 293 kb)

Supplementary Table 6

List of 645 bimodal genes (XLSX 76 kb)

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Lovatt, D., Ruble, B., Lee, J. et al. Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue. Nat Methods 11, 190–196 (2014). https://doi.org/10.1038/nmeth.2804

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