GSM5831258
mTECs were washed in wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine), bound to concavalin A beads (Bangs Laboratories), permeabilized in wash buffer plus 0.05% digitonin, and incubated overnight with 1:50 primary antibody (anti-H3K27ac [Abcam], -Hnf4α [Abcam], -Grhl1 [Novus], -Pou2f3 [Sigma], or -IgG [Rockland]) at 4°C in wash buffer plus 0.05% digitonin, 2mM EDTA and 0.1% BSA. The next day, H3K27ac, Grhl1, IgG, and some Pou2f3 samples were lightly fixed with 0.1% paraformaldehyde at room temperature for 2 minutes before proceeding. (We and others have observed improved CUT&Tag signal-to-noise ratio for some TFs with light fixation.) Samples were incubated with secondary antibody (guinea pig anti-rabbit [Rockland] for H3K27ac, Grhl1, Pou2f3, and IgG; rabbit anti-mouse [Abcam] for Hnf4α) at 1:100 for 1 hour at room temperature in wash buffer plus 0.05% digitonin. Samples were washed and incubated with pA-Tn5 (Addgene #124601, gift from Steve Henikoff, purified in-house) at 1:200 for 1 hour at room temperature in 300mM NaCl wash buffer plus 0.01% digitonin. Samples were washed twice then tagmented for 1 hour at 37°C in 300mM NaCl wash buffer plus 0.01% digitonin and 10mM MgCl. Tagmentation was halted with EDTA, sodium dodecyl sulfate and proteinase K for 1 hour at 55°C. Tagmented DNA was phenol-chloroform extracted and amplified by PCR with NEBNext 2X Master Mix (NEB) using the following program: 72°C for 2min, 98°C for 30s, 16 cycles of 98°C for 10s and 63°C for 10s, 72°C for 1min, hold. Amplified libraries were quantified by Qubit (Thermo) and Tapestation (Agilent).