PCR cycling conditions
Steps	Temperature	Time	Cycles
Gap filling	72°C	5 min	1
Initial denaturation	95°C	30 s	1
Denaturation	95°C	10 s	∗
Annealing and extension	63°C	10 s
Final extension	72°C	1 min	1
Hold	8°C	Infinite

^∗Optimal number of cycles for the “Denaturation” and “Annealing and extension” steps is determined at step 55 and should be adapted here. As mentioned in Figure 10 legend, during the actual library preparation step, the starting concentration of the CUT&Tagged genomic DNA sample is 5 times to that of the starting concentration in the qPCR system in step 54. So, the optimized cycle numbers obtained in step 55 should be reduced by 2 cycles for actual library preparation PCR.