The thymus of 6-week-old mice was dissected and minced with a razor. Thymic fragments were pipetted up and down in 1 mL of Hepes-RPMI (Wako). The supernatant was used as lymphocytes, and the remaining fragments were digested by incubation in RPMI 1640 medium containing 0.05 U/mL Liberase (Roche) and 0.01% w/v DNase I (Sigma-Aldrich) at 37°C for 12 min x3 times after each incubation and Pipetting. Reactions were stopped by adding an equal volume of FACS buffer containing 1 mM ethylenediaminetetraacetic acid and washing with FACS buffer. Total cell count was calculated as the sum of lymphocytes and digested cells. Single-cell emulsions were obtained using a 10x Genomics Controller and a v2 Library and Gel Bead kit (10X Genomics). RNA-sequencing libraries were prepared according to the 10x 3′ v2 kit protocol.