We estimated the inversion efficiency using a PCR-based approach as follows. First, a DNA segment of the non-mutagenic or the mutagenic allele was amplified by PCR using F7 and R7 primers (Figure 4—figure supplement 2A). The PCR products were purified using a Wizard SV PCR and Gel Purification kit (Promega, Madison, WI) and digested with BglI. Via this digestion, the PCR products specific to the mutagenic allele (M1 and M2 bands; Figure 4—figure supplement 2B) were separated from those specific to the non-mutagenic allele upon gel electrophoresis (N1 and N2 bands; Figure 4—figure supplement 2B). The intensity of each band was quantified using ImageJ software, and the value was normalized by the size (bp) of the band, providing the relative mole quantity (q) of DNA molecules in each band. The inversion efficiency was obtained as the percentage of the ratio of the sum of the relative mole quantity of M1 and M2 (qM1 + qM2) to that of all DNA bands (qM1 + qM2 +qN1 + qN2). Examples of the estimated inversion rate and its correlation to shha expression levels are shown in Figure 4—figure supplement 2C.