EdU-PLA Nascent DNA Protein Detection

EdU-PLA to detect proteins at nascent DNA was performed as described (Taglialatela et al., 2017 (link)), with minor modifications. Briefly, cells were pulsed with 10 μM EdU for 10 min cells before being permeabilised with nuclear extraction buffer (10 mM PIPES, 20 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100). Where appropriate, cells were exposed to 4 mM HU for 5 hr before pre-extraction. Cells were then fixed with 3.6% paraformaldehyde for 10 min at room temperature and blocked with ADB (Antibody Dilution Buffer; 3% BSA in PBS) overnight at 4°C. EdU was conjugated to biotin by incubating cells in Click reaction buffer for 1 hr at room temperature containing 10 μM Diazo-biotin Azide, 10 mM sodium ascorbate, and 1 mM copper (II) sulfate in PBS. Following the Click reaction, cells were blocked in ABD before incubated in primary antibodies before proceeding with proximity ligation using a Duolink Detection Kit in combination with anti-Mouse PLUS and anti-Rabbit MINUS PLA Probes (Sigma Aldrich) according to the manufacturer’s instructions. Cells were imaged as below and the number of PLA signals per nuclei quantified.