Junction PCR and sequencing

Genomic DNA was extracted with lysis buffer (10 mM Tris-HCl, pH 8.2, 200 mM NaCl, 5% SDS solution, 200 μg/mL proteinase K and 10 mM EDTA) from either individual or pools of 72 hpf zebrafish embryos and then used to PCR amplify the 5’ and 3’ junction fragments of target genes or the region flanking the loxP site using the appropriate primers (Supplementary file 7). The PCR products were either directly sent for sequencing (F₁ and F₂ embryos) or cloned into pMD18-T (TAKARA) for clonal sequencing (F₀ embryos).