Cut&Tag of Treg cells from spleen of mus
The library preparation for CUT&Tag was performed according to the manufacturers instruction with Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme, TD901). In brief, 1105 sorted GFP+ Treg cells were washed with 500 l wash buffer. Cell pellets were resuspended in 100l wash buffer and activated concanavalin A-coated magnetic beads were added and incubated for 10min at room temperature. Cell-bound beads were then collected and resuspended in 50 l of antibody buffer containing primary antibody (BAF60b antibody, provided by Prof. Zhuoxian Meng, Zhejiang University School of Medicine; RUNX1 antibody, Abcam, ab198977; Normal IgG, Cell Signaling Technology, 2729), and incubated at 4 overnight. After removing the primary antibody, the secondary antibody (Donkey anti-rabbit IgG, Abcam, Ab96921) diluted at 1:50 in 50l of Dig-wash buffer was added and incubated at room temperature for 1h. The cells were then incubated with 0.04m Hyperactive pG-Tn5 Transposase diluted in 100l of Dig-300 buffer at room temperature for 1h. Standard tagmentation and amplification were performed according to the instructions. Finally, amplified DNA libraries were purified with VAHTS DNA Clean Beads (Vazyme, N411) and sequenced on an Illumina NovaSeq platform, and 150-bp paired-end reads were generated. Raw reads were filtered with fastp (version 0.23.2) and aligned to the mm10 mouse genome using Bowtie2 (version 2.3.5.1) with standard alignment parameters. Apparent PCR duplicates were marked with Picard Mark Duplicates function and removed for downstream analysis. Genome browser tracks were generated using deepTools with RPKM normalization (version 3.5.1). The tracks were visualized using the Integrated Genome Viewer. Peaks were called using MACS2 (version 2.2.7.1) with the following parameters:  -f BAMPE -q 0.001 --keep-dup all. The genomic distribution of peaks were annotated with ChIPseeker (version 1.28.3). Heatmaps and read density plots of genomic signal density were generated using Deeptools.