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Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq

Rebboah, Elisabeth and Reese, Fairlie and Williams, Katherine and Balderrama-Gutierrez, Gabriela and McGill, Cassandra and Trout, Diane and Rodriguez, Isaryhia and Liang, Heidi and Wold, Barbara J. and Mortazavi, Ali (2021) Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210429-100236539

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Abstract

Alternative RNA isoforms are defined by promoter choice, alternative splicing, and polyA site selection. Although differential isoform expression is known to play a large regulatory role in eukaryotes, it has proved challenging to study with standard short-read RNA-seq because of the uncertainties it leaves about the full-length structure and precise termini of transcripts. The rise in throughput and quality of long-read sequencing now makes it possible, in principle, to unambiguously identify most transcript isoforms from beginning to end. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here, we develop and characterize long-read Split-seq (LR-Split-seq), which uses a combinatorial barcoding-based method for sequencing single cells and nuclei with long reads. We show that LR-Split-seq can associate isoforms with cell types with relative economy and design flexibility. We characterize LR-Split-seq for whole cells and nuclei by using the well-studied mouse C2C12 system in which mononucleated myoblast cells differentiate and fuse into multinucleated myotubes. We show that the overall results are reproducible when comparing long- and short-read data from the same cell or nucleus. We find substantial evidence of differential isoform expression during differentiation including alternative transcription start site (TSS) usage. We integrate the resulting isoform expression dynamics with snATAC-seq chromatin accessibility to validate TSS-driven isoform choices. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells that can be further quantified with companion deep short-read scRNA-seq from the same cell populations.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.04.26.441522DOIDiscussion Paper
ORCID:
AuthorORCID
Rebboah, Elisabeth0000-0003-2273-0189
Reese, Fairlie0000-0002-9240-0102
Williams, Katherine0000-0001-8061-0105
Balderrama-Gutierrez, Gabriela0000-0002-5794-4518
Trout, Diane0000-0002-4928-5532
Wold, Barbara J.0000-0003-3235-8130
Mortazavi, Ali0000-0002-4259-6362
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted April 27, 2021. We would like to thank Melanie Oakes at UC Irvine Genomics High-Throughput Facility (GHTF) for her help with PacBio sequencing. This work was supported in part by grants from the National Institutes of Health (UM1HG009443) to A.M. and B.J.W. The authors have declared no competing interest.
Funders:
Funding AgencyGrant Number
NIHUM1HG009443
Record Number:CaltechAUTHORS:20210429-100236539
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210429-100236539
Official Citation:Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq. Elisabeth Rebboah, Fairlie Reese, Katherine Williams, Gabriela Balderrama-Gutierrez, Cassandra McGill, Diane Trout, Isaryhia Rodriguez, Heidi Liang, Barbara J. Wold, Ali Mortazavi. bioRxiv 2021.04.26.441522; doi: https://doi.org/10.1101/2021.04.26.441522
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108866
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Apr 2021 17:39
Last Modified:29 Apr 2021 17:39

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