Validating Genome Editing in Tumor Cells

To test whether the genome-editing techniques actually altered the genome in tumor cells, we used FACS to sort GFP⁺ tumor cells for genomic DNA isolation for genotyping and for RNAs to verify the expression of oncogenes. RNAs were isolated with the RNeasy micro kit (Qiagen), and cDNA was synthesized according to a previously described method58 (link). RT-PCR for MYC, EGFR/EGFRvIII, and TBP was done with the primers listed in Supplementary Table 4. Genomic DNAs were isolated with DNeasy Blood & Tissue kits (Qiagen) according to the manufacturer’s instruction. The CRISPR–Cas9-targeted genome loci of tumor-suppressor genes were amplified using the primers listed in Supplementary Table 5. The PCR products were inserted into T vector (Promega) according to the manufacturer’s instructions. Ninety-six colonies per gene were cultured for sequencing.