USA)
USA). 2.2. profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting Goat polyclonal to IgG (H+L)(HRPO) at interrelations. and 4 C. Nuclei pellets were resuspended in 5 mL chilled PBS made up of 1% BSA and 0.2 U/L RNase Isosilybin A inhibitor and cell debris was removed by a final filtration step. After centrifugation for 8 min at 600 and 4 C, the supernatant was carefully removed and nuclei were resuspended in 3 mL Nuclei PURE storage buffer. The samples were transferred to cryotubes, snap-frozen in liquid nitrogen, and stored at ?80 C until processing. Sequencing was conducted by Genewiz (Leipzig, Germany) around the 10xGenomics system (Carlsbad, CA, USA). Single nuclei were captured in droplet emulsions and snRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3 Isosilybin A Gel Bead and Library V3 Kit (Carlsbad, CA, USA). RNA was controlled for sufficient quality on an Agilent 2100 Bioanalyzer system (Santa Clara, CA, USA) and quantified using a Qubit Fluorometer (Waltham, MA, USA). Libraries were subsequently sequenced around the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA. USA). 2.2. Computational Data Analysis The snRNA-seq fastq data files were aligned with kallisto (v.0.46) to the generated mm10 genome (Ensembl release 98) index. The UNIX source code made up of the detailed actions of the generation is provided at our FairdomHub/iRhythmics instance (https://doi.org/10.15490/fairdomhub.1.study.713.1). Additionally, the latest version of the complete index build was shared at Zenodo for further reuse (https://doi.org/10.5281/zenodo.3623148). This index contains the spliced and unspliced transcript annotations of the mm10 murine needed for RNA velocity analysis. The kallisto alignment files were subsequently quantified with bustools (v.0.39.3) as previously described [8]. Subsequently, transcripts were integrated into R by using the BUSpaRse R-package (v.0.99.25) to be able to use the downstream processing tool Seurat (v.3.1.1). Isosilybin A For clustering, dimensionality was initially reduced by principal component analysis and numbers of the most variable principal components were selected using heuristic methods implemented in Seurat. For an improved UMAP clustering and identification of small subgroups, we included the upstream processing algorithm harmony (v.1.0) [9]. The RNA velocity was conducted with the velocyto R-package (v.0.6) [6]. Sets of well-known marker genes were used to assign the underlying cell types of the generated clusters, as summarized in our computational R script. In addition, novel cell cluster markers recently identified by other groups working with single-nucleus data4 were applied and found to be transferable to our dataset. The detailed experimental protocol, computational scripts, top 100 transcripts per cluster as well as the expression of the top markers for our identified clusters can be accessed from FairdomHub/iRhythmics. Raw data is provided in the Single Cell Expression Atlas via Arrayexpress (Accession ID: E-MTAB-8751). 3. Results and Discussion Single-nucleus analysis included a total of 8635 nuclei and 22,568 genes in which each cell exhibits an average total expression of 2662.6 reads. The analysis revealed 24 distinct clusters as a UMAP representation showing a global connectivity among the groups (Physique 1). The largest clusters can be attributed to populations of endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) containing ~2500, ~2200, and ~2000 nuclei, respectively. Open in a separate window Physique 1 Single-nucleus transcriptome characteristics of pooled whole Fzt:DU mice hearts (= 4). UMAP clustering of snRNA-seq data (8635 nuclei) reveals 24 distinct.