Replicate 1 of Top1 CUT&Tag in HCT116, add ZnTTAPc
CUT&Tag was constructed as described (Kaya-Okur et al., 2019) with some modifications. 10^5 cells were washed twice with 100 ??L wash buffer (20 mM HEPES-KOH at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1?? protease inhibitors), and then resuspended with 100 ??L wash buffer. 10 ??L pre-activated concanavalin A coated magnetic beads (Novoprotein, Shanghai) were added to the cells and incubated on a rotator for 10 minutes at room temperature. The supernatant was removed and bead-bound cells were resuspended in 100 ??L dig-wash buffer (0.05% digitonin, 0.1% BSA, 20 mM HEPES-KOH at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 2 mM EDTA, 1?? protease inhibitors) containing 9 ?g/mL recombinant anti-DNA:RNA hybrid antibody (S9.6, Abcam) or 1 ?g/mL recombinant anti-Topoisomerase I antibody (ab109374, Abcam). After one-hour rotation at room temperature, the cells were washed twice with 100 ??L of dig-wash buffer and resuspended with 100 ??L dig-wash buffer containing 1 ??L Goat anti-mouse IgG, incubated for another one hour at room temperature in the dark, and washed three times with dig-wash buffer. A 1:250 dilution of pA-Tn5 adapter complex (~0.2 ??M) was prepared in 100 ??L dig-wash buffer and added to the cells followed by one-hour rotation at room temperature. After five washes with dig-wash buffer, the bead-bound cells were resuspended with 50 ??L of tagmentation buffer (20 mM HEPES-KOH at pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 1?? protease inhibitors) and incubated at 37??C for one hour. To terminate the tagmentation reaction, 2.5 ??L of 0.5 M EDTA, 2.5 ??L of 10% SDS and 1 ??L of 20 mg/mL proteinase K were added and incubated at 55??C for 30 min and then for 15 minutes at 65??C. The DNA was extracted with 122 ?L Ampure XP beads for library preparation. DNA was eluted in 40 ?L of 5 mM Tris-HCl (pH 8.0). To amplify libraries, 21 ??L DNA was mixed with a universal i5 and a uniquely barcoded i7 primer, and amplified with NEB Q5 high-fidelity 2?? master mix. The libraries were purified with 1.2?? volume of Ampure XP beads (Beckman Counter), and libraries were eluted in 30 ?L 10 mM Tris-HCl (pH 8.0).