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

研究目的：本研究旨在探究整合素β3（integrin beta3, Itgb3）缺陷对骨髓细胞基因表达的调控作用。
实验方法：采用预冷的含2%胎牛血清的磷酸盐缓冲液（phosphate buffered saline, PBS）冲洗Apoe(-/-)、Itgb3(+/+)及Apoe(-/-), Itgb3(-/-)小鼠的股骨与胫骨以收集骨髓细胞。使用Lympholyte（Cedarlane, CL0531）通过密度梯度离心法分离骨髓细胞：以1300 g离心20分钟后，收集界面处的细胞，用无钙镁离子汉克斯平衡盐溶液（Hank's Balanced Salt Solution, Ca²+/Mg²+-free HBSS）洗涤，随后通过70 μm尼龙网过滤制备为单细胞悬液。后续开展10X Genomics单细胞3’端RNA测序文库构建与测序实验。单细胞RNA测序数据采用Cell Ranger v3.1.0进行处理，将测序reads比对至修饰版小鼠转录组mm10，选取Cell Ranger筛选出的优质细胞条形码用于后续数据分析。
实验结果：对来自Apoe(-/-)、Itgb3(+/+)及Apoe(-/-), Itgb3(-/-)三种基因型小鼠骨髓中分离的不同细胞类型的转录组表达谱进行了对比分析。
研究结论：Apoe(-/-)、Itgb3(+/+)及Apoe(-/-), Itgb3(-/-)三种基因型小鼠的骨髓细胞中，基因表达谱存在显著差异。
整体实验设计：从Apoe(-/-)、Itgb3(+/+)及Apoe(-/-), Itgb3(-/-)三种基因型小鼠中收集骨髓细胞，进行10X Genomics单细胞3’端RNA测序文库构建与测序。基于经典的细胞类型特异性标记基因对鉴定得到的细胞簇进行注释。