Single-cell RNA-Seq of entire ventricles for C57BL/6J and B6.Rag2del mice after cardiomyocyte transplantation

We compared the cellular and molecular outcome following cardiomyocyte transplantation in wildtype and T- and B cell deficient Rag2del mice. Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. 8-12 weeks old, male and female C57BL/6J mice (Charles River, Wilmington, MA, USA) and B6.Rag2del mice (Jackson laboratory, Bar Harbor, ME, USA) were used for this study. Ventricles were harvested from neonatal transgenic B6.eGFP mice, the tissue was manually minced, and single cell suspensions were prepared using the Pierce primary cardiomyocyte isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers protocol. Entire mouse ventricles were isolated and enzymatically digested as described above. Cells were then labelled with CD45 magnetic beads (Miltenyi Biotec, Germany) and positively enriched using the AutoMACS instrument (Miltenyi Biotec, Germany). Viable macrophages/monocytes (CD45+CD11b+CD11c-DAPI-Lactadherinlo), dendritic cells (CD45+CD11b+CD11c+ DAPI-Lactadherinlo), and NK cells (CD45+CD11b-CD11c+NK1.1+ DAPI-Lactadherinlo) were then sorted on the BD FACSAriaTM IIIu (Becton Dickinson, Franklin Lakes, NJ, USA) roughly in a 1:1:1 ratio into DMEM containing 10% FCS before processing for 10× Genomics single-cell RNA sequencing (scRNA-Seq). Libraries for scRNA-Seq were constructed according to the 10×?Genomics protocol using the GemCode Single-Cell 3' Gel Bead and Library V3 Kit. Quality of amplified cDNA and final libraries were evaluated on the 2100 Bioanalyzer instrument (Agilent) using a High Sensitivity NGS Analysis Kit (Advanced Analytical). Subsequent sequencing was conducted on the HighSeq4000 Sequencing System using the HiSeq SBS and HiSeq PE Cluster Kit V4 (all Illumina, San Die-go, CA. USA).