ATAC-sequencing of young and middle-aged SSPCs from mouse bone. ATAC-sequencing of young and middle-aged SSPCs from mouse bone

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. To identify transcriptional changes that occur with aging we performed bulk RNA sequencing on young (3 month) and middle-aged (12 month) SSPCs. Overall design: Tibial and femoral SSPCs were isolated by centrifugation. Bone marrow was resuspended in growth media (GM) [DMEM containing 10% FBS and 1% penicillin/streptomycin; and then plated in 150cm2 tissue- culture flasks. Media changed the following day and then every two days. All cellular assays were performed with SSPCs at passage one from at least three different mice and young and middle-aged SSPCs for ATAC-sequencing.

骨骼干细胞与祖细胞（Skeletal stem and progenitor cells, SSPCs）介导骨骼的稳态维持与损伤修复。随机体衰老，此类细胞生成的成骨细胞减少、脂肪细胞增多，最终导致骨骼完整性丧失。驱动这一有害表型转变的分子机制目前仍未完全阐明。为鉴定衰老过程中发生的转录组改变，我们对3月龄年轻小鼠与12月龄中年小鼠的骨骼干细胞与祖细胞开展了批量RNA测序（bulk RNA sequencing）。整体实验设计：通过离心法分离胫骨与股骨来源的骨骼干细胞与祖细胞，将获取的骨髓重悬于生长培养基（growth media, GM）中，该培养基为添加10%胎牛血清（fetal bovine serum, FBS）与1%青霉素-链霉素的DMEM培养基；随后将细胞接种至150cm²的组织培养瓶中。次日更换培养基，之后每两日更换一次。所有细胞实验均使用至少3只不同小鼠来源的第1代骨骼干细胞与祖细胞开展；针对转座酶可及性测序（ATAC-sequencing），我们分别使用年轻与中年小鼠的骨骼干细胞与祖细胞进行实验。