TY - JOUR
T1 - Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes
AU - Kannan, Suraj
AU - Miyamoto, Matthew
AU - Lin, Brian
AU - Zhu, Renjun
AU - Murphy, Sean
AU - Kass, David
AU - Andersen, Peter
AU - Kwon, Chulan
N1 - Publisher Copyright:
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 4.0 International license.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2019/5/30
Y1 - 2019/5/30
N2 - Rationale Single cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Objective We investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. Methods and Results We found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties. Conclusions Our study enables high quality dissection of adult CM biology at single-cell resolution.
AB - Rationale Single cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Objective We investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. Methods and Results We found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties. Conclusions Our study enables high quality dissection of adult CM biology at single-cell resolution.
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U2 - 10.1101/654954
DO - 10.1101/654954
M3 - Article
AN - SCOPUS:85093577310
JO - Advances in Water Resources
JF - Advances in Water Resources
SN - 0309-1708
ER -