title: GSM7528377: WT4; Mus musculus; OTHER
library_construction_protocol: Lymphocytes were isolated from the spleen and lymph nodes of 6- to 8-week-old Foxp3RFP/∆2Foxp3GFP-creR26YFP female mice and enriched for CD4+ T cells using magnetic beads. Then, CD4+YFP+RFP+ (WT Tregs) and CD4+YFP+RFP- (Foxp3∆2 tTregs) cells were sorted to a typical purity of >95%. CUT&Tag libraries were generated with the Hyperactive In-Situ ChIP Library Prep Kit for Illumina Kit (Vazyme Biotech, TD901). Briefly, 50,000 cells for each CUT&Tag library were incubated with 10 μl balanced concanavalin A-coated magnetic beads at RT for 10 min. The bead-bound cells were resuspended with 50 μl of antibody buffer. In all, 1 μg anti-Foxp3 antibody (Abcam, ab150743) was then added to the bead-bound cells. After 2-h incubation at RT, the primary antibody was discarded carefully and 0.5 μl goat anti-rabbit IgG (Vazyme, Ab206-10-AA) diluted with 50 μl Dig-wash buffer was added to the cells. The cells were then incubated at RT for 1 h. Afterward, samples were washed gently with 800 μl Dig-wash buffer, and 0.58 μl pG–Tn5 together with 100 μl Dig-300 buffer was added to the samples. After 1-h incubation at RT, samples were washed gently with 800 μl Dig-300 buffer. In all, 300 μl tagmentation buffer was then added to each sample and the samples were incubated at 37 °C for 1 h. The interactions were quenched by adding 10 μl 0.5 M EDTA, 3 μl 10% SDS, and 2.5 μl 20 mg/ml Proteinase K and incubating the samples at 50 °C for 1 h. Phenol–chloroform and ethanol precipitation, as well as PCR and size selection, were performed as described in Hi-C library construction. All CUT&Tag libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode subsequently.