Aged ATA tissues were enzymatically digested for 60 min at 37°C in a solution containing type II collagenase (10 mg/mL; Sigma Aldrich, C6885) and elastase (1 mg/mL; Worthington Biochemistry, LS002292). Digested tissues were filtered through a 70 μm cell strainer to obtain single-cell suspensions. Viable cells were enriched using a Dead Cell Removal Kit (Miltenyi Biotech, 130-090-101). Cell viability was assessed by Trypan Blue staining (0.4%; Bio-Rad, 1450013) using a TC20 automated cell counter (Bio-Rad). Approximately 15,000 viable cells per sample were loaded onto a 10x Genomics Chromium Controller to generate gel beads-in-emulsion (GEMs). Single-cell 3′ gene expression libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v2 (10x Genomics; PN-120237, PN-120236, PN-120262) according to the manufacturer's instructions.