title: Illumina HiSeq 4000 sequencing; Single-cell RNA-Seq of entire ventricles for C57BL/6J and B6.Rag2del mice after cardiomyocyte transplantation
design: Single-cell RNA-Seq of entire ventricles for C57BL/6J and B6.Rag2del mice after cardiomyocyte transplantation
library_construction_protocol: 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. Prior to use, mice were bred in the animal facility of the Rostock University Medical Centre and maintained in specified pathogen-free conditions with food and water ad libitum. Following surgical procedures, the mice were allowed to recover in individual cages. 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). Briefly, cells were resuspended in PBS with 0.04% BSA and diluted to a concentration of 1,000 cells/μl. Cell viability amounted > 91% as assessed using the Cellometer Auto 2000 Cell Viability Counter. Single cells were then captured in droplet emulsions using the GemCode Single-Cell Instrument (10× Genomics) with a target output of 2,500 cells. Libraries for scRNA-Seq were constructed according to the 10× Genomics protocol using the GemCode Single-Cell 3′ Gel Bead and Library V3 Kit.