Droplet Digital PCR Protocol Design

Primers and probes for ddPCR were designed using Primer3 software^{91 (link)}. Primer specificity was tested using standard PCR on knock-in clones to yield only unique products. Sanger sequencing confirmed product identity. ddPCR was performed on a Biorad QX200 system according to the manufacturer’s recommendations. ddPCR Supermix without dUTP was used for amplification. Dual-labeled probes were synthesized by Microsynth (Germany) and contained either FAM or HEX at the 5′-end and BHQ1 quencher at the 3′-end. Annealing/elongation temperature was set to 60.8 °C. Five units of BstY1 restriction enzyme (NEB) per 20 µl reaction was added directly to the reaction mix. Sequences of primers and probes as well as final probe concentrations are listed in the Supplementary Tables 1 and 5. Data were analyzed by QuantSoft software (www.bio-rad.com).