Single-cell RNA sequencing of human bone marrow monocytes post-myocardial infarction at acute and chronic timepoints
Single-cell RNA sequencing of human bone marrow monocytes post-myocardial infarction at acute and chronic timepoints
Sternal BM was collected during surgical procedures. Inclusion criteria for MI patients included those scheduled for coronary artery bypass grafting (CABG) surgery, with a history of MI (acute: < 3 months after MI or chronic:  > 3 months after MI) and a left ventricular EF greater than 45%. The described criteria were also applied to the control group, with the exception that a history of MI was not present. The samples were subjected to Ficoll separation and subsequently frozen. On the day of sorting, the samples were thawed, and a total of 3,000 - 20,000 human bone marrow monocytes (Lineage negative, CD45 positive, HLA-DR positive) were sorted in 2% BSA in PBS in two biological replicates. 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).