title: GSM9206294: R397W_Treg_2 (Foxp3 CUT&Tag); Mus musculus; OTHER
library_construction_protocol: Cells were fixed with 0.1% formaldehyde for 2 minutes at 25 °C. Fixation was quenched with 75 mM glycine. Fixed cells were washed once with wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail). Spike-in nuclei (Active Motif, 53168) were added to the cell suspensions and washed once with wash buffer. Concanavalin A-conjugated beads (EpiCypher, 21-1411) were activated with bead activation buffer (20 mM HEPES, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂) and incubated with cells for 10 minutes at room temperature. Bead–cell mixtures were incubated with 0.5 µg anti-Foxp3 rabbit polyclonal antibody (in-house) or normal rabbit IgG (CST, 2729S), together with antibody against spike-in nuclei (Active Motif, 53168), in antibody buffer (wash buffer supplemented with 0.01% digitonin, 2 mM EDTA, 0.1% BSA) overnight at 4 °C on a rotator. Mixtures were then incubated with anti-rabbit IgG (Rockland, 611-201-122) in wash buffer supplemented with 0.01% digitonin for 30 minutes at room temperature on a rotator. Bead–cell mixtures were next incubated with pAG-Tn5 (EpiCypher, 15-1017) in wash buffer supplemented with 0.01% digitonin and 150 mM NaCl (final 300 mM NaCl) for 1 hour at room temperature on a rotator. Tagmentation was carried out by incubating bead–cell mixtures with tagmentation buffer (wash buffer supplemented with 0.01% digitonin, 150 mM NaCl, and 10 mM MgCl₂) for 1 hour at 37 °C with shaking (600 rpm). Samples were treated with 16.5 mM EDTA and 0.1% SDS, vortexed, and incubated overnight at 65 °C with shaking (900 rpm) for de-crosslinking. Proteinase K was then added, followed by incubation for 1 hour at 50 °C with shaking (900 rpm). DNA was extracted using phenol–chloroform–isoamyl alcohol (PCIA). Tagmented DNA were amplified by PCR with 14-15 cycles using barcoded primers