RNA Extraction and Quality Assessment

Following expansion, 3 × 10⁵ CB Tregs, CB Tconv, APB Tregs, and APB Tconv were lysed in DNA/RNA lysis buffer (Zymo Research) and stored at −80°C. RNA extraction was achieved using ZR-Duet^TM DNA/RNA MiniPrep (Zymo Research, Catalog No. D7001), per the manufacturer’s instructions. Quality assessment of RNA was achieved by Experion^TM Automated Electrophoresis System (BIO-RAD) using Experion RNA High Sensitivity Reagents and Experion Standard Sensitivity RNA chips following the manufacturer’s protocol. Only samples with a minimum RNA concentration of 10 ng/μL and RNA Quality Index (RQI) ≥9.4 were used.