Hypertrophic chondrocytes and osteogenesis in P6 mouse tibia

It has been shown that hypertrophic chondrocyte (HC) can become osteoblast, contributing to the formation of trabecular bone and endosteum. However, it is unclear what genes regulate the process of differentiation from chondrocyte to osteoblast. Conditional knock-out of Irx 3 and Irx5 in HCs can reduce the trabecular bone amount and there is also some evidence that ablation of Irx3 and Irx5 in HCs can promote bone marrow adipogenesis. In order to investigate the gene regulatory network with different dosage of Irx3/5 in HC descendants (i.e. in Loss-of-Function mutant and heterozygous control), we collected HC descendant cells from proximal tibia of mice at postnatal day 6 and performed single cell RNA seq with 10X Genomics System to profile the gene expression pattern in HC descendants. We sequenced one sample. Cells were harvested from proximal and distal tibia. The sample (a pool of two tibia from the same male P6 mouse) is on Col10a1Cre;Rosa26-Tdtomato;Irx3 +/Δ;Irx5 +/- (Irx3/5 heterozygous control). Cells with or without Tdtomato fluoresence are digested out by sequential digestion, enriched by sequential centrifugation and resuspension and subject to standard 10X Genomics protocol.