Table 3. RADD sequential reaction conditions.

RADD is performed in two sequential reactions without aspirating reagents between reactions. The lesion processing mix (Left) is placed on prepared tissues and placed in a humidified incubator. The gap filling mix (Right) is added directly to the lesion processing mix and incubated for an additional hour. The reagents are then aspirated and the cells are washed and incubated with anti-digoxigenin antibody.

Lesion processing mix	100 μL total reaction volume	Gap filling mix	100 μL total reaction volume
UDG (NEB M0280s)	2.5 U	Klenow exo- (Thermo Fisher EP0422)	5 U
FPG (NEB M0240L)	4 U	Digoxigenin dUTP (Sigma Aldrich 11573152910)	1 μM
T4PDG (NEB M0308S)	5 U	Therm Pol buffer (NEB B9004S)	10 μL
EndoIV (NEB M0304L)	5 U
hAAG (NEBM0313S)	5 U
NAD+ (100x, NEB B9007S)	500 μM
BSA (Sigma Aldrich)	200 μg/mL
Therm Pol buffer (NEB B9004S)	10 μL