Extended Data Fig. 3: Related to Fig. 3.

a–j, 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. iT_reg cells were also sorted from the mLN for RNA-seq. a, Gating strategy used for sorting of CD4⁺ T cells in sequential order. Note that this gating strategy was used in all FACS data shown in this study. b, Sorting strategy of the indicated populations. CD8α and Tomato expression among CD4⁺ CD8β^Lo T cells (left) from IE (top) and mLN (bottom). ThPOK and neuropilin-1 expression among Tomato⁺CD8α^– (red), Tomato⁺CD8α⁺ (purple) and Tomato^–CD8α⁺ (green) cells from IE (top) and mLN (bottom). c, Levels of ThPOK expression in each sorted population in the IE. d, Mapped accessible chromatin regions relative to promoters of all genes in ATAC-Seq data (top) and their relative read counts per genomic regions (bottom). e, ATAC-Seq peak annotations as indicated per cell type. f, Percent of total differentially accessible chromatin regions as follows: 5′UTR and promoters (Promoter; red), 3′UTR with exons and introns (Gene Body; blue), transcriptional termination site (TTS; gray) and intergenic (black). g, Euclidean distance correlation of chromatin accessibility profiles of all samples. h, Volcano representation of differentially expressed genes between IE iT_reg (higher expression in blue) and mLN iT_reg cells (higher expression in red), performed by Wald pairwise comparison test, p_adj < 0.05 values were considered significant. i, T_reg cell signature from clusters 1- 3 and IEL signature from cluster 5 of the bulk RNA-seq heatmap (Fig. 3d) overlaid onto the scRNA-Seq UMAP from Fig. 1. j, Curated list of gene set enrichment analysis determined using the fgsea package with p_adj < 0.05 of Hallmark pathways (black), chemical genetic perturbations pathways (white), and immune pathways (Retinoic acid (RA) pathways; green, Interferon pathways; blue, CD8-program; gray, and T_reg cell program; silver). Significant differentially accessible chromatin regions p_adj < 0.01(d–g) and significant differentially expressed genes p_adj < 0.05 in RNA-Seq (h-j). 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 (d–g). Each sample for RNA-Seq (d-f) consisted of 650 or 800 cells per mouse, n = 2 or 3 mice over 2 independent experiments (h–j).