Extended Data Fig. 4: Related to Fig. 4.

a–c, iFoxp3^TomThPOK^GFP mice were treated with tamoxifen for 10 weeks and induced T_reg cells (iT_reg; CD4⁺Tomato⁺GFP^Hineuropilin-1^–CD8α^–), pre-IELs (CD4⁺Tomato⁺ GFP^LoCD8α^–), exT_reg-IELs (CD4⁺Tomato⁺GFP^LoCD8α⁺), and CD4-IELs (CD4⁺ Tomato^–GFP^LoCD8α⁺) were sorted in bulk from the IE. Assay for transposase-accessible chromatin (ATAC) or RNA libraries were prepared followed by sequencing of indicated populations. a, Pearson’s correlation (r) of log2 fold changes of RNA-Seq vs ATAC-Seq of total ATAC peaks averaged (top) or at promoters only (bottom) of indicated cell types. P value (p) for r coefficient as indicated. b, ATAC-Seq peaks of indicated populations and ThPOK chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of in vivo differentiated Foxp3⁺ splenic T_reg cells displayed on the integrative genomics viewer (IGV) in select regions as indicated. Overlap regions between ChIP- and ATAC-Seq indicated on bottom. c, Numbers of differentially expressed genes (DEGs, n = 2 or 3 per cell type) (silver for increase, gray for decrease) in between cell types in sequential progression as performed by Wald pairwise test as indicated, with differentially accessible chromatin regions (DACR, n = 2 per cell type) within those genes (red for increase, blue for decrease in accessibility). Significant DACR p_adj < 0.01 and significant DEG p_adj < 0.05 in RNA-Seq. Each sample for ATAC-Seq was from n = 9 (1^st) or n = 6 (2^nd) pooled mice over 2 independent experiments. 15,000 or 19,000 iT_reg cells, 30,000 or 17,000 pre-IELs, 12,000 or 11,000 exT_reg-IELs and 40,000 CD4-IELs were used. Each sample for RNA-Seq consisted of 650 or 800 cells per mouse, n = 2–4 mice over 2 independent experiments.