Ddpcr supermix for probes reagents

PDX cells were labeled with 6-carboxyfluorescein succinimidyl ester (Life Technologies) and sorted into single cells using a FACSAria™ III flow cytometer (BD Biosciences, CA, USA). Wells with a single green fluorescence signal were manually inspected and selected for amplification of genomic DNA with a GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Little Chalfont, UK). The mutant alleles were detected using ddPCR Supermix for Probes reagents (Bio-Rad, Hercules, CA, USA) implemented using a QX200 ddPCR system, following the manufacturer’s protocols. The negative signal of droplets was normalized with a vehicle control, and the numbers of wild-type or mutation alleles in droplets were estimated in a Poisson distribution. Variant allele frequency was calculated by counting copies of mutation alleles over the total number of detected alleles. We regarded genotypes of detected variants as homozygous when the variant allele frequency was higher than 90 %. Sequences of the primers used in ddPCR are available in Additional file 6.

ddPCR was performed based on the water−oil emulsion droplet technology, using the ddPCR™ Supermix for Probes reagents in the QX200™ Droplet Digital™ PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For quantification of AAV vector, the eluted cellular DNA was PCR amplified using Taqman set targeting the SaCas9 AAV transgene and as a reference rhesus macaque TERT gene (Supplementary Table S1). A total of 50−100 ng DNA from each tissue was used as template for ddPCR amplifications with thermal cycling conditions used 98 °C 5 min, 45 cycles (98 °C 5 min), 45 cycles (98 °C 15 s, 60 °C 30 s). For quantification of AAV vector expression 1 μl of RT-PCR reaction was used and the same SaCas9 Taqman set like for DNA analysis, as a reference 0.1 μl of reaction was used and Taqman set specific to beta-actin. Data acquisition and analysis are done using QX200 droplet reader and QuantaSoft™ software provided with the instrument.

Droplet digital PCR was performed on the QX200 droplet digital PCR system (BioRad). Five μL of the cfDNA isolated as described above was used for ddPCR and all samples were analyzed in triplicate. Validated kits for both the wild type and the T790M-mutated EGFR were purchased from BioRad (cat #10031250 and cat #10031247) and the assay was performed with the ddPCR Supermix For Probes Reagents (BioRad, cat #186–3025). Results were analyzed with the QuantaSoft software (Bio-Rad).