High throughput single-cell RNA sequencing of intact adult cardiomyocytes and non-myocytes using a split-pool approach

MOTIVATIONAdult cardiomyocytes are difficult to profile by whole-cell single-cell RNA sequencing because of their large size and fragility, which make them poorly compatible with standard workflows. Current approaches for adult cardiomyocyte transcriptomics often require a trade-off between data quality and throughput, thus, studies instead rely heavily on sequencing of nuclei alone. Therefore, we set out to develop a high-quality and scalable workflow for adult heart cells using in-cell ligation and split-pool barcoding strategies to address this methodological gap. This workflow may be further generalisable to other large cell types or samples containing cell populations with highly unequal RNA content. SUMMARYAdult cardiomyocytes are difficult to profile by whole-cell single-cell RNA sequencing (scRNA-seq). Here, we developed a high-quality and scalable workflow for adult heart cells using in-cell ligation and split-pool barcoding. We identified per-cell RNA content as a significant variable that must be accounted for. Separation of cardiomyocytes (large cells) and non-cardiomyocytes (small cells) before library construction, and allocation of deeper sequencing to cardiomyocytes, produced high-quality whole-cell datasets for both compartments. Compared with single-nucleus RNA sequencing, whole-cell cardiomyocyte profiling better recovered metabolic, mitochondrial, cytoplasmic translational, and contractile gene programs. This workflow provides a practical method for scalable, high-quality cardiomyocyte whole-cell scRNA-seq and offers general strategies for other large cell types or samples containing cell populations with highly unequal RNA content.