Single cell RNA-seq to delineate the gene expression of bone marrow cells

Purpose: The goal of this study is to investigate the role of integrin beta3 (Itgb3) deficiency on the gene expression of bone marrow cells. Methods: Bone marrow was harvested by flushing the femurs and tibias of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) mice in PBS with 2% fetal bovine serum on ice. Cells were isolated from bone marrow by density gradient centrifugation using Lympholyte (Cedarlane, CL0531). After centrifugation at 1300 g for 20 min, bone marrow cells were collected from the interface and washed in Ca2+/Mg2+ - free HBSS and dissociated to a single-cell suspension by filtering through a 70-µm nylon mesh. The cells were then subjected to 10X Genomics single cell 3' RNA-seq libraries construction and sequencing. scRNA-seq data was processed with Cell Ranger v 3.1.0. Reads were aligned to a modified version of the mouse transcriptome mm10. The top cell barcodes selected by Cell Ranger were utilized for downstream analysis. Results: Expression profiles of the transcriptome are compared between cell types isolated from the bone marrow of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) mice. Conclusions: Significant differences in the expression profile of genes are present in bone marrow cells of Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) genotypes. Overall design: Bone marrow cells were harvested from Apoe(-/-), Itgb3(+/+) and Apoe(-/-), Itgb3(-/-) mice and subjected to 10X Genomics single cell 3' RNA-seq library construction and sequencing. Identified cell clusters were annotated based on classical cell type-specific markers.