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Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types

Ishaan Gupta, Paul G Collier, Bettina Haase, Ahmed Mahfouz, Anoushka Joglekar, Taylor Floyd, Frank Koopmans, Ben Barres, August B Smit, Steven Sloan, Wenjie Luo, Olivier Fedrigo, M Elizabeth Ross, Hagen U Tilgner
doi: https://doi.org/10.1101/364950
Ishaan Gupta
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Paul G Collier
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Bettina Haase
2The Rockefeller University, 1230 York Avenue, New York, NY, 10065
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Ahmed Mahfouz
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
3Leiden Computational Biology Center, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden
4Delft bioinformatics Lab, Delft University of Technology, van Mourik Broekmanweg 6, 2628 XE Delft
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Anoushka Joglekar
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Taylor Floyd
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Frank Koopmans
5Dept. Molecular and Celullar Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam, The Netherlands
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Ben Barres
6Department of Neurobiology, Stanford University, 299 Campus Dr, Stanford, CA 94305-5125
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August B Smit
5Dept. Molecular and Celullar Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, Amsterdam, The Netherlands
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Steven Sloan
6Department of Neurobiology, Stanford University, 299 Campus Dr, Stanford, CA 94305-5125
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Wenjie Luo
7Brain and Mind Research Institute and Appel Alzheimer’s research institute, Weill Cornell Medicine
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Olivier Fedrigo
2The Rockefeller University, 1230 York Avenue, New York, NY, 10065
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M Elizabeth Ross
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Hagen U Tilgner
1Brain and Mind Research Institute and Center for Neurogenetics, Weill Cornell Medicine, 413 east 69th Street, New York, NY 10021
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Abstract

Full-length isoform sequencing has advanced our knowledge of isoform biology1–11. However, apart from applying full-length isoform sequencing to very few single cells12,13, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success14,15, but these methods do not describe full-length mRNAs. Single cell short-read 3’ sequencing has allowed identification of many cell sub-types16–23, but full-length isoforms for these cell types have not been profiled. Using our new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) we determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. We elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites6–9,24,25, which for individual molecules requires long reads. We produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).

ScISOr-Seq describes isoforms from >1,000 single cells from bulk tissue without cell sorting by leveraging two technologies in three steps: In step one, we employ microfluidics to produce amplified full-length cDNAs barcoded for their cell of origin. This cDNA is split into two pools: one pool for 3’ sequencing to measure gene expression (step 2) and another pool for long-read sequencing and isoform expression (step 3). In step two, short-read 3’-sequencing provides molecular counts for each gene and cell, which allows clustering cells and assigning a cell type using cell-type specific markers. In step three, an aliquot of the same cDNAs (each barcoded for the individual cell of origin) is sequenced using Pacific Biosciences (“PacBio”)1,2,4,5,26 or Oxford Nanopore3. Since these long reads carry the single-cell barcodes identified in step two, one can determine the individual cell from which each long read originates. Since most single cells are assigned to a named cluster, we can also assign the cell’s cluster name (e.g. “Purkinje cell” or “astrocyte”) to the long read in question (Fig 1A) – without losing the cell of origin of each long read.

Footnotes

  • ↵& author, who tragically passed away and could not see the result of this work.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 08, 2018.
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Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types
Ishaan Gupta, Paul G Collier, Bettina Haase, Ahmed Mahfouz, Anoushka Joglekar, Taylor Floyd, Frank Koopmans, Ben Barres, August B Smit, Steven Sloan, Wenjie Luo, Olivier Fedrigo, M Elizabeth Ross, Hagen U Tilgner
bioRxiv 364950; doi: https://doi.org/10.1101/364950
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Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types
Ishaan Gupta, Paul G Collier, Bettina Haase, Ahmed Mahfouz, Anoushka Joglekar, Taylor Floyd, Frank Koopmans, Ben Barres, August B Smit, Steven Sloan, Wenjie Luo, Olivier Fedrigo, M Elizabeth Ross, Hagen U Tilgner
bioRxiv 364950; doi: https://doi.org/10.1101/364950

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