Replicate 1 of DSB CUT&Tag in HeLa, add PyBI
CUT&Tag was constructed as described (Zheng et al., 2024) with some modifications. The cell bound magnetic beads were first permeated with 100 uL 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 uL protease inhibitors) for 30 min, and then the supernatant was discarded and added to 100 uL TdT buffer (200 mM potassium cacodylate, 25 mM Tris, 0.01% (v/v) Triton X-100, 1 mM CoCl2 (pH 7.2)) containing 5 uM biotin-11-dUTP and 5 U TdT to label the intracellular DNA break site for 1h. The bead-bound cells were resuspended in 100 uL dig-wash buffer containing 10 ug/mL recombinant anti-Biotin antibody (ab201341, Abcam).