GSM9437266: BoneRANKL-fm, fractured, index2; Mus musculus; RNA-Seq
Tibiae were isolated at post-fracture day 14 and minced using scissors or tweezers. The fragmented bones were digested in DMEM containing Collagenase P, Liberase TH, and DNase I at 37 °C with gentle shaking for 1 h. Dissociated cells were collected every 20 min and passed through a 100 μm nylon mesh filter. Cells were pelleted by centrifugation and resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA). Red blood cells were lysed using 0.165 M ammonium chloride. The resulting single-cell suspension was stained with antibodies against CD45.2, Ter119, and CD31. Fixable viability dye (eBioscience) was used to stain dead cells. CD45.2-Ter119-CD31-tdTomato+ cells were sorted for single-cell 3′ gene expression library preparation. Library construction was performed according to the manufacturer's instructions (Single Cell 3' v3.1 protocol, 10x Genomics), with slight modifications.  Cells were directly sorted into the master mix and loaded, together with partitioning oil and gel beads, into the chip to generate gel bead-in-emulsions (GEMs).