After the visualization of 50 metaphases spreads and 50 nuclei with the combined DNA-RNA FISH for each biological replicate, the slides were delicately washed two times in PBS 1X at room temperature and then treated with RNase A solution (2XSSC, 0,1 mg/ml of RNase A enzyme, Thermo Fisher Scientific) for 1 h at 37 °C. Three washes in 70%, 90%, and 100% ethanol were performed and then the slides were stained with VECTASHIELD Antifade Mounting Medium with DAPI (Vector laboratories),
For RNAse H treatment, after counting FISH spots the slides were washed two times in PBS 1X at room temperature and treated with RNase H solution (PBS 1X, 10% Reaction Buffer 10X and 0.05 U/µL of RNase H enzyme, Thermo Fisher Scientific) for 1 h at 37 °C. As before, three washes in 70%, 90%, and 100% ethanol were performed and the slides were stained with VECTASHIELD Antifade Mounting Medium with DAPI (Vector laboratories). To further verify the effect of RNase H on FISH signals, RNase H solution was also used for 1 h at 37 °C to treat the slides before the three washes in 70%, 90%, and 100% ethanol and the combined DNA-RNA FISH protocol.
For RNAse H treatment, after counting FISH spots the slides were washed two times in PBS 1X at room temperature and treated with RNase H solution (PBS 1X, 10% Reaction Buffer 10X and 0.05 U/µL of RNase H enzyme, Thermo Fisher Scientific) for 1 h at 37 °C. As before, three washes in 70%, 90%, and 100% ethanol were performed and the slides were stained with VECTASHIELD Antifade Mounting Medium with DAPI (Vector laboratories). To further verify the effect of RNase H on FISH signals, RNase H solution was also used for 1 h at 37 °C to treat the slides before the three washes in 70%, 90%, and 100% ethanol and the combined DNA-RNA FISH protocol.