Single cell sequencing allows for an in-depth characterization of complex tissues and their cell-type composition. 1 However, there are two major issues when it comes to the cardiovascular system (i) the difficulty of dissociating the adult mammalian heart tissue without damaging constituent cells and (ii) technical limitations regarding cell capture techniques leading to an underrepresentation of individual cell types (ie cardiomyocytes) due to their large cell size and irregular shape. 2 To avoid these shortcomings, we desisted from single-cell RNA-seq and conducted single-nuclei RNA-seq. For most studies inbred mouse lines are used. However, human populations are genetically diverse, hence inbred lines may be inappropriate, when considering the genetic contribution to disease. Therefore, we provide essential transcriptome data for entire adult murine hearts at the cellular level for both, an inbred strain (C57BL/6NRj) and an outbred strain (Fzt: DU). We isolated nuclei from whole hearts of age-matched adult mice after cervical dislocation. Mice were handled in accordance to Directive 2010/63/EU on the protection of animals. Sequencing was performed on a 10Â Genomics system and subsequent downstream analysis conducted with Seurat, including the preprocessing algorithm harmony for batch correction. 3 Moreover, RNA velocity analyses served to characterize transcription kinetics in identified cell types hinting at their specific cellular inter-relations. 4 The detailed experimental protocol, computational script, and top 100 markers for the identified clusters can be accessed from FairdomHub/iRhythmics. The raw data are available at Arrayexpress (E-MTAB-8751, E-MTAB-8848). Here, we present a comparative sequencing analysis in entire adult mammalian hearts, providing realistic cell-type distributions combined with RNA velocity kinetics. Our integrated single-nuclei analysis included a total of 12 099 nuclei (8635 Fzt: DU—blue, 3464 BL6—orange) as well as 22 568 genes and revealed 23 distinct clusters as an UMAP representation for both datasets (Figure 1). The largest nuclei clusters can be attributed to populations of cardiomyocytes, fibroblasts, and endothelial cells containing

$3700, $3300, and $2900 nuclei, respectively. In murine hearts, binucleation in cardiomyocytes has to be considered and in comparison to former sequencing analyses using single cells 1 the amount of cardiomyocytes in our current analysis was up to two-fold higher (ie 39% of BL6 nuclei belong to cardiomyocytes meaning that $20% of total cells belong to this lineage), reflecting that we could surpass the isolation and sizespecific effects of this cell type. When viewing the individual data sets, it was evident that the proportion of cardiomyocytes in the BL6 is higher than in Fzt: DU. In contrast, there are in total two times more endothelial cells in Fzt: DU than in BL6 mice. However, amongst these endothelial cells we found a population that comprises markers clearly related to cardiomyocyte function (eg Gja1, Atp2a2, Ttn, Ryr2, Myh6—complete list at FairdomHub/iRhythmics). Our additional RNA velocity analysis utilized information about nascent mRNA as a predictor for future cell states and allowed to study transcription kinetics that are indicated as arrows in Figure 1. The results support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype confirming previous findings. 5 BL6 mice seem to lack this intermediate cell population but have more conventional cardiomyocytes instead. Regarding the endothelial cells, our data further contradict earlier flow cytometry studies that suggest …