design: The library preparation for ATAC-Seq was performed according to the manufacturers instruction with TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501). Briefly, 5104 sorted GFP+ Treg cells were lysed in lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, 0.1% (v/v) Igepal CA-630) for 5min on ice. The nuclei pellet was resuspended in 50L TruePrep Tagment Enzyme buffer and then incubated at 55 for 10min. Purify the transposition product with VAHTS DNA Clean Beads (Vazyme, N411). PCR was then performed to amplify the library for 14 cycles using the following PCR cycles: 72 for 3min; 98 for 30s, followed by thermocycling at 98 for 15s, 60 for 30s, and 72 for 40s, and finally 5min at 72. After PCR, libraries were purified with VAHTS DNA Clean Beads. The libraries were sequenced on an Illumina NovaSeq platform, and 150-bp paired-end reads were generated. The pair-end reads were trimmed using fastp(version 0.23.2). After quality control, the reads were mapped to the mm10 mouse genome using Bowtie2(version 2.3.5.1). The unmapped reads, mitochondrial reads and nonuniquely mapped reads were removed by SAMtools (version 1.10). PCR duplicates were removed by Picard MarkDuplicates (version 2.25.0). Bigwig tracks were generated using deepTools with RPKM normalization (version 3.5.1). Integrated genomics viewer was used to display coverage tracks. The read coverage across all genomic regions was calculated by deepTools "computeMatrix" function. The read density at each region was visualized by "plotHeatmap" function(deepTools). Peaks were identified by MACS2(version 2.2.7.1) with the following parameters: --nomodel extsize 200 --shift 100. Differential peak analysis between samples was performed using Homer program get Differential Peaks.