TGFB_H2AK119ub1  (CUT&TAG)
In brief, 5 x 105 cells washed twice with 1.0 mL of wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and 1 protease inhibitors]. Concanavalin A-coated magnetic beads (10 l; Cell Signaling Technology, 93569) were activated and incubated with the cells at room temperature for 10 minutes. After removing the supernatant, the bead-bound cells were resuspended in 100 l of antibody buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1 protease inhibitors, 0.05% digitonin, 0.01% NP-40, and 2 mM EDTA]. Subsequently, 1.65ug H2AK119ub antibody (Diagenode, C15410002) was added, and the mixture was incubated overnight at 4C with rotation. After two washes with dig-wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1 protease inhibitors, 0.05% digitonin, 0.01% NP-40], the samples were incubated with 1.5ug mouse anti-rabbit IgG (Sigma, R2655) in 100 l of antibody buffer at room temperature for 1 hour. Bead-bound cells were washed once with 150 l of dig-wash buffer to remove unbound antibodies. Secondary rabbit anti-mouse IgG antibody (1.5ug; Thermo, 31188) diluted 1:100 incubated for another hour, followed by three washes in dig-wash buffer. The pA-Tn5 adapter complex (Cell Signaling Technology, 79561) was diluted 1:25 in dig-300 buffer [20 mM HEPES (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, 1 protease inhibitors, 0.05% digitonin], and with the bead-bound cells with 50 l diluted pA-Tn5 incubated at room temperature for 1 hour with rotation. Cells were then washed three times in dig-300 buffer to remove unbound pA-Tn5. Tagmentation was performed by resuspending cells in 40 l of tagmentation buffer [10 mM TAPS-KOH (pH 8.3), 10 mM MgCl, 10% DMF] and incubating at 37C for 1 hour. The reaction was stopped by adding 1.5 l of 0.5 M EDTA, 0.5 l of 10% SDS, and 1 l of proteinase K (20 mg/ml; Roche), followed by incubation at 55C for 1 hour and 75 C for 20 min. Tagmentation products were purified with 1.8x AMPure XP beads (Beckman,20745200) and eluted in 10 l of 0.1% Tween 20. The eluent was mixed with 10 U of Bst 2.0 WarmStart DNA polymerase (NEB, M0538) and 0.4 U Q5 high-fidelity DNA polymerase (NEB, M0491L) in 1x Q5 polymerase reaction buffer, incubated at 65C for 30 minutes to conduct the strand displacement reaction, and heat-inactivated at 80C for 20 minutes. Sequencing libraries were generated by amplifying with a universal i5 primer and a unique i7 primer using Q5 high-fidelity DNA polymerase. Libraries were size-selected with 0.5 SPRIselect beads (Beckman, B23318) and validated by Agilent TapeStation analysis before Illumina sequencing.