GSM9351993: RAD51D tile 3 replicate #3 passage 0; synthetic construct; OTHER
Genomic DNA from P0 and P2 cell populations was isolated using CleanNA Blood and Tissue magnetic bead kit (Bulldog Bio) To amplify integrated RAD51D mutant libraries, “outer PCR” was performed on a 500 ng gDNA template with primers flanking the RAD51D cDNA using the PrimeSTAR GXL kit (Takara), with 16 replicate PCR reactions for each gDNA sample to maintain library complexity. To quantify mutation frequency in sorted cells, mutant libraries were subjected to deep amplicon tile sequencing as described (Hemker et al, 2025). Briefly, 1 µL of outer PCR library product was amplified in a limited (≤10) cycle PCR, using primers directed at constant bases flanking the targeted tile within the RAD51D cDNA. Primer 5' tails contained constant sequencing adaptor stub sequences. Each resulting product was then diluted and further amplified to add sequencing adaptors and sample-specific unique dual 10 bp index sequences.