title: Single-cell RNA sequencing of mouse myeloid cardiac tdTomato+ and tdTomato- cells (Fgd5-CreERT2) post-myocardial infarction upon DMSO and 4-oxo-RA treatment.
design: Single-cell RNA sequencing of mouse myeloid cardiac tdTomato+ and tdTomato- cells (Fgd5-CreERT2) post-myocardial infarction upon DMSO and 4-oxo-RA treatment.
library_construction_protocol: In Fgd5CreERT2 mice, LAD was performed, followed by a 2-day treatment with either vehicle (DMSO) or 4-oxo-RA. On day 3 post-MI, hearts were extracted, and dTomato-positive and dTomato-negative myeloid cells (CD11b pos) were isolated from the myocardium. Cardiac myeloid cells were sorted in 2% BSA in PBS in 1 biological replicate. n.a. Sorted cells in PBS supplemented with 2% BSA were pelleted at 300g for 5 min at 4 °C and washed once in ice-cold PBS containing 0.5% BSA. Cells were resuspended in PBS with 0.5% BSA to a maximum cell concentration of 390 cells/_l and placed on ice. Cell viability was assessed by trypan blue staining. A cell suspension aliquot (2 _l) was diluted 1:2 in 0.4% trypan blue solution, incubated for 2 minutes on a microscope slide and analyzed under a microscope. All samples for scRNA-seq analysis showed more than 90% viable cells. Single cell RNA-sequencing was performed on the 10X Genomics platform using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 dual index (10x Genomics, PN-1000268) following the manufacturer's instructions. A total of 43 _l of cell suspension was loaded onto a chip G according to the manufacturer's instructions, aiming for a targeted cell recovery of up to 10,000 cells. PCR cycles for cDNA and final library generation were adjusted according to the target cell recoveries.  The quality of the obtained cDNA and final libraries was assessed by capillary electrophoresis (Fragment analyzer, HS NGS Fragment Kit, Agilent).