replicate 1 of NCL CUT&Tag in HeLa-S3
The G4-CUT&Tag method was constructed following the described protocol (Zheng et al., 2024) with some modifications. Briefly, a total of 10^5 cells were washed twice with 100 uL 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 µL wash buffer. Next, 10 uL of pre-activated concanavalin A coated magnetic beads were added to the cells and incubated on a rotator for 10 minutes at room temperature. The supernatant was removed, and the bead-bound cells were resuspended in 100 uL of 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 either 250 nM of recombinant G4P or 0.7 ug/mL of recombinant anti-NCL antibody (14574, CST). After two-hours rotation at room temperature, the cells incubated with G4P were washed twice with 100 ul of dig-wash buffer and resuspended with 100 uL of dig-wash buffer containing 1 uL of anti-FLAG M2 antibody (F1804, Sigma), incubated for another one hour at room temperature. The buffer containing primary antibodies was then removed from the cells, and 100 uL of dig-wash buffer containing 1 uL of Goat anti-mouse IgG (AP124, Sigma) or 1 uL of Goat anti-rabbit IgG (AP132, Sigma) was mixed with the cells and incubated for one hour at room temperature. After washing three times with dig-wash buffer, a 1:250 dilution of pA-Tn5 adapter complex (~0.2 uM) was prepared in 100 uL of dig-wash buffer and added to the cells, which were then rotated for one hour at room temperature. After five washes with dig-wash buffer, the bead-bound cells were resuspended with 50 ul 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 ul of 0.5 M EDTA, and 1 ul of 20 mg/mL proteinase K were added and incubated at 55°C for 30 min. The DNA was then extracted using 120 ul of Ampure XP beads (Beckman Counter) for library preparation. DNA was eluted in 40 ul of 5 mM Tris−HCl (pH 8.0). To amplify the libraries, 16 ul of DNA was mixed with a uniquely barcoded i5 and a uniquely barcoded i7 primer, and amplified using Q5 high-fidelity 2× master mix (NEB). The libraries were purified with 1.3× volume of Ampure XP beads, and eluted in 20 ul of 10 mM Tris−HCl (pH 8.0).