Single-cell RNA-Seq of porcine peripheral blood monocytes

We performed 10×?Genomics single-cell RNA sequencing on a mixed population of CD3+ PBMCs isolated from three healthy Large White pigs. Considering that cell resolution of scRNA-seq analysis can be improved based on large datasets, we also downloaded 7 pbulic PBMC scRNA-seq datasets using the same technique (10×?Genomics). After rigorous quality control and the removal of batch effects, a total of 14,595 transcriptomes were obtained from 34,220 cells in 8 PBMC samples for subsequent analyses. Using the graph-based uniform manifold approximation and projection (UMAP) clustering algorithm in Seurat package, we identified 14 major cell types (32 clusters) from peripheral blood circulating cells of pigs, and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8+ T cells, we defined 4 CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Through single-cell regulatory network inference, we identified key regulatory factors during CD8 T cell differentiation. Moreover, cell-cell communication analysis underscored extensive intercellular interactions among different cell types. Lastly, our cross-species analysis highlighted both similarities and potential differences between species. Overall design: Peripheral blood mononuclear cells (PBMCs) were collected from 3 healthy Large White pigs at the experimental pig farm of Huazhong Agricultural University. Peripheral blood samples (2 mL) were collected into EDTA anticoagulant tube and gently inverted 2-3 times to ensure thorough mixing. Subsequently, the mixed blood samples were transferred to 15 mL centrifuge tube and treated with 6 mL of red blood cell lysis solution for 15 min, followed by centrifugation at 1600 rpm for 10 min at 4? to collect cell pellets. The cell pellets were then re-suspended in cold phosphate-buffered saline (PBS), passed through a 100 µm cell strainer, and incubated with 3 µL CD3e antibody (clone BB23-8E6-8C8, BD Pharmingen) for 30 min at 4? in the dark, followed by FACS to obtain living CD3+ PBMCs. The cell viability assay using trypan blue staining. After cell counting, the samples from the three healthy pigs were mixed in equal volumes to obtain 9×105 cells. The mixed cells were used for constructing 10× genomics sequencing library and subsequent sequencing by Genergy Biotechnology (Shanghai, China)