library_construction_protocol: ImmGen standard protocols (http://www.immgen.org) for cell preparation and sorting were followed to generate data for all conditions – IL2/JES6-1, IL2 immuno-cytokine treatment/IL2 anti-FITC controls or PBS control. Whole spleens were harvested and homogenized through 70um filter and centrifuged before red blood cells were removed using ACK lysis buffer for 3 minutes on ice and centrifugation. Remaining cells were washed once with 5mL of FACS buffer (Phenol red-free DMEM, 2% FBS, 0.1% azide, and 10 mM Hepes, pH 7.9) and then resuspended in FACS buffer to be stained for 20 minutes on ice with antibodies for flow cytometry. Stained cell suspensions were sorted once for ATACSeq or twice for RNASeq to obtain pure samples of T regulatory cells. Splenic cell suspensions from FOXP3-IRES-GFP mice were stained on ice for 20minutes with anti-CD4, anti-TCRb+, anti-CD19, anti-CD8a, anti-CD11c, NK1.1, and propidium iodide (PI) as a viability dye. Cells were sorted as PI-Dump [CD19-CD8a-CD11c-NK1.1]-CD4+TCRb+GFP+]. Nuclei isolation, transposition, GEM generation, and library construction targeting capture of 10000 cells were carried out as detailed in the Chromium Next GEM Single Cell ATAC manual (10x Genomics) with modifications to allow sample hashtagging (see below). Libraries were pooled and sequenced on an Illumina NovaSeq 6000 to a final median depth of approximately 20-30,000 paired-end reads per cell. Sequencing data were converted to fastq files, aligned to the mm10 reference genome, and quantified per cell using Cell Ranger ATAC software (10x Genomics, v2.1). To include multiple experimental conditions in the same scATAC run, we hashtagged cells using a modification of the ASAP-seq strategy (Mimitou et al., 2021) for low cell input primary cell samples. Before sorting, cells were hashtagged with mouse TotalSeqA DNA-barcoded hashtag antibodies at the same time as staining with fluorophore-conjugated antibodies (BioLegend). Cells were sorted into RPMI + 5% FCS in DNA Lo-Bind tubes (Eppendorf, cat # 022431021). After spinning down for 5 min at 500g in a refrigerated centrifuge at 4°C, cells were resuspended in 100 μl chilled 0.1x Omni Lysis buffer (1x Omni Lysis buffer (10mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40 substitute/IGEPAL, 0.01% Digitonin, 1% BSA in nuclease free water) diluted 1:10 in Wash/Lysis Dilution Buffer (10mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 1% BSA in nuclease free water), gently mixed by pipetting, and incubated on ice for 6.5 min. Following lysis, 100 μl chilled wash buffer was added and gently mixed by pipetting. Cells were spun down for 5 min at 500g at 4°C, all but 5 μl of supernatant was removed, and 45 μl of chilled 1x nuclei buffer (10x Genomics) was added without mixing. After one more centrifugation step at 500g, 4°C for 5 min, supernatant was removed, and samples were resuspended in 7ul 1x nuclei buffer for cell counting and input into transposition, barcoding, and library preparation according to the Chromium Next GEM Single Cell ATAC manual (10x Genomics). Modifications to the original 10X protocol were made as described in the original ASAP-seq publication (Mimitou et al., 2021) and as detailed at https://citeseq.files.wordpress.com/2020/09/asap_protocol_20200908.pdf. Briefly, 0.5 μl of 1uM BOA bridge oligo was spiked into the barcoding reaction. During GEM incubation, an additional 5 min incubation at 40°C was added to the beginning of the protocol. 43.5 instead of 40.5 μl of Elution Solution I was added during silane bead elution to recover 43 μl. 40 μl was used for SPRI clean up as indicated in the protocol, while 3 μl was set aside. During SPRI cleanup, the supernatant was saved. The bead bound fraction was processed as in the protocol, while for the supernatant fraction, 32 μl SPRI was added for 5 min. Beads were collected on a magnet, washed twice with 80% ethanol, and eluted in 42 μl EB. This 42 μl was combined with the 3 μl set aside from the previous step as input into the HTO indexing reaction. HTO Indexing PCR was run with partial P5 and indexed Rpxx primers (https://citeseq.files.wordpress.com/2020/09/asap_protocol_20200908.pdf) as: 95°C 3 min, 12-14 cycles of (95°C 20 sec, 60°C 30 sec, 72°C 20 sec), 72°C 5 min. The PCR product was cleaned up with 1.6X SPRI purification for quantification and sequencing alongside ATAC libraries.