Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

Abstract

RationaleSingle 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).\n\nObjectiveWe investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs.\n\nMethods and ResultsWe 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.\n\nConclusionsOur study enables high quality dissection of adult CM biology at single-cell resolution.