Single-cell RNA sequencing of bone marrow cells and spleen isolated from mice after sham surgery or myocardial infarction, followed by treatments (vehicle/4-oxo-RA)
Single-cell RNA sequencing of bone marrow cells and spleen isolated from mice after sham surgery or myocardial infarction, followed by treatments (vehicle/4-oxo-RA)
Myocardial infarction was induced in female C57BL/6J mice aged 6 to approximately 12 weeks through permanent occlusion of the left anterior descending artery (LAD). Mice were intraperitoneally injected on the 1st and 2nd days after MI surgery with either 30 mg/kg body weight 4-oxo-RA (Sigma-Aldrich; MI+at-RA condition), or with the corresponding amount of DMSO in phosphate-buffered saline (PBS) (MI+vehicle condition). The mice were euthanized for analysis 3 days after MI. Murine bone marrow (BM) and spleen cells were isolated from spleen, the femur, tibia, hip bone and vertebrae by gentle crushing with mortar and pestle in PBS. Red blood cell lysis was performed with ACK Lysing Buffer (Thermo Fisher Scientific) for 5_min at room temperature (RT). Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen) was used for lineage negative enrichment according to the manufacturerÕs protocol. Briefly, the BM was stained with 1:4 dilution of the Lineage Cocktail for 30Ð60_min at 4_¡C on a rotating wheel. Labelled cells were then incubated for 20_min with 400_µl of washed Dynabeads coated with polyclonal sheep anti-rat IgG per sample. Depletion of lineage cells was performed using a magnet. Lineage-depleted BM cells were stained with lineage markers (Gr1, CD11b, B220, Ter119, CD4 and CD8a), ckit, Sca1, CD150, CD48 and CD34. Sorting was then performed using a FACS Aria II, III or FACSymphony (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany. Each 25,000 LSK cells (Lin-negative, Sca1-positive, c-Kit-positive) of sham+vehicle, MI+vehicle or MI+ 4-oxo-RA mice were sorted into 2% BSA in PBS, respectively, in 2 biological replicates. n/a For scRNA-seq analysis, 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 3Õ 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 manufacturerÕs 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).