amplicon seq for sgRNA target sites and the scar sequence for AttP- tandem GPC-CRISPR
HEK293T cells harboring the AttP-tandem GPC-CRISPR gene circuit were treated with gibberellin A4 and abscisic acid for sequential mutation of APC, MLH1, and SMAD4. 200~300 base pairs amplicon flanking the sgRNA target sites were amplified to detect indel mutations. 150~200 base pairs amplicon flanking the scar sequence consisting of the recombinase recognition sites were also sequenced to detect cells at specific stage within cascade. The amplicon was prepped for NGS using Illumina Truseq Library Prep kit v2.