replicate 1 of IgG CUT&Tag in HeLa
The CUT&Tag method was constructed following the described protocol (Chen et al., 2025) with some modifications. A total of 10^5 cells were washed twice with 100 uL wash buffer (20 mM HEPESKOH at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1 protease inhibitors). Next, 10 uL of pre-activated concanavalin A coated magnetic beads were added to the cells in 100 uL wash buffer 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 HEPESKOH at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 2 mM EDTA, 1 protease inhibitors) containing 250 nM of G4P, 200 nM of CK13, 2 uL of anti-NCL antibody (ab70493, Abcam), or 1 uL of anti-Top1 antibody (ab109374, Abcam), respectively. After 2 hours rotation at room temperature, the cells incubated with G4P or CK13 were washed twice with 100 uL of dig-wash buffer and resuspended with 100 uL of dig-wash buffer containing 2 uL of anti-FLAG M2 antibody (Sigma-Aldrich), or 2 uL of anti-V5 tag antibody (ab15828, Abcam), incubated for another 1 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-Aldrich) or 1 uL of Goat anti-rabbit IgG (AP132, Sigma-Aldrich) was mixed with the cells and incubated for 1 hour at room temperature. After washing three times with dig-wash buffer, a 1:100 dilution of pAG-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 1 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 HEPESKOH (pH 7.5), 150 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 1 protease inhibitors) and incubated at 37  for 1 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  for 30 minutes. 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 TrisHCl (pH 8.0). To quantify the library DNA, 2 uL of extracted DNA was mixed with an i5 and an i7 primer and amplified using Q5 High Fidelity 2 Master Mix (New England Biolabs) containing 1SYBR green I. Real-time quantitative PCR was performed on CFX Duet Real-Time PCR System (Bio-Rad). The cycling condition was 72  for 5 minutes, 95  for 3 minutes, and followed by 40 cycles of 30 s at 95  and 30 s at 65 . 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 (New England Biolabs). The PCR cycles for G4P and CK13 groups were 16 to 18, and those for Top1, NCL, and IgG groups were 15, 17, and 22, respectively. The libraries were purified with 1.3  volume of Ampure XP beads and eluted in 20 uL of 10 mM TrisHCl (pH 8.0).
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