Nucleic Acid Manipulation for Immunofluorescence

For RNase A treatment or RNase H treatment, the cells were permeabilized with 0.2% Triton X‐100 in PBS for 5 min at room temperature. Then 200 µg mL⁻¹ RNase A (NEB) or 100 U mL⁻¹ RNase H (NEB) was applied to the cells for 1 h at room temperature and washed three times with PBS. Then, the cells were fixed with 2% paraformaldehyde to IF or GST‐protein hybridization. For GST‐protein hybridization, 0.2 µg µL⁻¹ recombinant GST‐proteins were added to the cells and incubated for 1 h at room temperature. After washing with PBS, the cells were then stained with the GST and γH2AX antibodies. For pre‐rRNA blocking, 1 µg µL⁻¹ of pre‐rRNA was pre‐incubated with 0.2 µg µL^‐1 GST‐proteins for 30 min at room temperature and then subjected to the GST‐protein hybridization process.