Quantsoft software

The premixtures of TaqMan probes and primers consisted of 5 μM of TaqMan probe, 18 μM of a forward primer and 18 μM of a reverse primer. To optimize PCR annealing temperatures and characterize detection efficiencies, we mixed Bio-Rad ddPCR Supermix for Probes (12.5 μl), FAM- and HEX probe and primer premixture (1.25 μl each), and mixture of a genomic DNA (100 ng) and a knock-in allele plasmid (0.5 pg or series of diluted plasmid) in 25 μl total volume. Droplet generation, PCR reaction and droplets read were performed by a QX100 Droplet Generator, a C1000 Thermal Cycler and a QX100 Droplet Reader (Bio-Rad), respectively according to the instructions from the manufacturer. Droplets were analysed by QuantSoft software (Bio-Rad). The optimal annealing temperatures for LMNA, HIST2H2BE, CBX1 and PRKACA were 62, 60, 60, 59 and 58 °C, respectively.
To measure the knock-in efficiency, we mixed Bio-Rad ddPCR Supermix for Probes (12.5 μl), FAM- and HEX probe and primer premixture (1.25 μl each), and HEK293FT genomic DNA (100 ng) in 25 μl total volume. After droplet generation and PCR reaction, the knock-in frequencies were calculated by taking concentration ratio of a mutant-allele-specific FAM probe and a wild-type-allele-specific HEX probe (TUBA1A genomic locus).

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).