Assessment of Foxp3 and GFP antibodies for cellular staining and CUT&RUN-seq. (A) Schematic binding of Foxp3 and GFP antibodies in Treg cells isolated from Foxp3^gfp knock-in mice expressing an N-terminal GFP:Foxp3 fusion protein. (B) Comparison of Foxp3 and GFP antibodies with flow cytometric staining of Treg cells isolated from Foxp3^gfp mice. Cells were permeabilized with or without fixation before antibody staining. Data represent more than three experiments. (C) Cross-comparison of the CUT&RUN-seq results generated with rTreg and aTreg cells from Foxp3^gfp mice and with anti-GFP and anti-Foxp3 (FJK-16s) antibodies. Two replicates per condition are shown. (D) Foxp3 peaks at the Il10 locus in rTreg and aTreg cells revealed by CUT&RUN-seq using anti-GFP and anti-Foxp3 (FJK-16s) antibodies as described above. Arrowheads indicate increased Foxp3 binding in aTreg cells. Data represent two replicates. (E) Co-immunoprecipitation of Ets1 and Foxp3 in in vitro induced Treg cells. (F) A heatmap showing Foxp3 and Ets1 binding in Treg cells after CRISPR deletion of Ets1. NC, non-targeting negative control sgRNA. (G) DNA sequence motifs for transcription factors enriched at the regions with reduced Foxp3 binding (P < 0.05) after Ets1 CRISPR knockout (sgEts1) in Treg cells. (H) Foxp3 and Ets1 peaks at the Ikzf4 locus in Treg cells that received retroviral sgEts1 or sgNC. Data are representative of two replicates. Source data are available for this figure: SourceData FS1.