GSM8987143: Y; Mus musculus; RNA-Seq
The periosteal tissue was dissociated using a mixed enzyme solution prepared according to the manufacturer's protocol (Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China). The solution contained 2 mg/ml type II collagenase, 0.5 mg/ml dispase, 50 U/ml DNase. After preheating the mixed enzyme solution at 37°C for 30 minutes, periosteal tissue was incubated with the enzyme mixture in a water bath at 37°C. The digestion process was repeated four times, each for 15 minutes. After each digestion, the suspension was filtered through a 40 μm mesh and neutralized with an equal volume of complete medium (α-MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin). The remaining tissue was subjected to a second round of digestion. At the end of the four digestions, the cell suspension was centrifuged at 300 g for 5 minutes. The pellet was resuspended in 1 mL of 1× erythrocyte lysis buffer for 5 minutes, washed with 10 mL of ice-cold PBS, and centrifuged again at 300 g for 5 minutes. The final cell pellet was resuspended in 50 μL of PBS. Viability was assessed using the AO/PI staining method and an automated cell counter, with cell viability confirmed to be >80%. The concentration of the cell suspension was adjusted to approximately 1000 cells/μL. Library preparation and sequencing were conducted by Majorbio Bio-Pharm Technology Co. Single-cell suspensions were loaded onto the Chromium Single Cell Controller (10x Genomics) to generate single-cell gel bead emulsions, following the manufacturer's guidelines (Chromium Single Cell 3ʹ v3.1). The Chromium Single-Cell 3' Gel Bead and Library V3 Kit was used for cDNA synthesis and amplification, and sequencing was carried out on an Illumina NovaSeq 6000 platform.