Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes1,2,3,4,5,6,7,8,9,10,11, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far12,13. Although single splicing events have been described for ≤200 single cells with statistical confidence14,15, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3′ sequencing enables the identification of cellular subtypes16,17,18,19,20,21, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites6,7,8,9,22,23. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.

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This work used the Genomics Resources Core Facility and owes special thanks to J. Xiang and A. Wan. This work was supported by start-up funds (Weill Cornell Medicine) and a Leon Levy Fellowship in Neuroscience to H.U.T. as well as an R01 from the National Institute of Neurological Disorders and Stroke (1R01NS105477) to M.E.R.

Author information

Author notes

    • Ben Barres


    • Ishaan Gupta
    •  & Paul G Collier

    These authors contributed equally to this work.


  1. Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, New York, New York, USA.

    • Ishaan Gupta
    • , Paul G Collier
    • , Ahmed Mahfouz
    • , Anoushka Joglekar
    • , Taylor Floyd
    • , M Elizabeth Ross
    •  & Hagen U Tilgner
  2. The Rockefeller University, New York, New York, USA.

    • Bettina Haase
    •  & Olivier Fedrigo
  3. Leiden Computational Biology Center, Leiden University Medical Center, Leiden, the Netherlands.

    • Ahmed Mahfouz
  4. Delft Bioinformatics Lab, Delft University of Technology, Delft, the Netherlands.

    • Ahmed Mahfouz
  5. Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam, the Netherlands.

    • Frank Koopmans
    •  & August B Smit
  6. Department of Neurobiology, Stanford University, Stanford, California, USA.

    • Ben Barres
    •  & Steven A Sloan
  7. Brain and Mind Research Institute and Appel Alzheimer's Research Institute, Weill Cornell Medicine, New York, New York, USA.

    • Wenjie Luo


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P.G.C., I.G., S.A.S. and H.U.T. devised the experiments. P.G.C., B.H., I.G., S.A.S., O.F. and W.L. performed the experiments. I.G., A.B.S. and H.U.T. devised the analyses. I.G., A.M., A.J., T.F., F.K. and H.U.T. performed the analyses. All of the authors discussed and interpreted the results throughout the project. I.G. and H.U.T. wrote the paper with inputs from all of the other authors. B.B., M.E.R. and H.U.T. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hagen U Tilgner.

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    Supplementary Figures 1–7

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    Life Sciences Reporting Summary

  3. 3.

    Supplementary Note 1

    Supplementary note detailing methodology of defining trustworthy alignments to genes and detection of novel isoforms

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    Complete Annotation of RNA Isoforms and their cell-type specific expression in the mammalian cerebellum from P1 mouse

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    Annotation of Novel RNA Isoforms, which are novel with respect to UCSC and RefSeq annotation, and their cell-type specific expression in the mammalian cerebellum from P1 mouse

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