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

The use of single cell RNA sequencing (scRNA-seq) remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Here, we investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. We found that LP-FACS readily outperforms conventional FACS for isolation of structurally 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. Overall design: Here, we include raw and processed data from two separate experiments performed to assess LP-FACS isolation of cardiomyocytes. For the multichamber study, we isolated single adult (~3 months old) CMs using LP-FACS from various chambers of the heart. For the live/fixed study, we isolated ventricular CMs both pre- and post-ethanol fixation via LP-FACS.