Dhsacp1000485

Mitochondrial DNA copy number analysis was performed by ddPCR as described previously by Memon et al. [51 (link)], using probes targeting the mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) (assay ID: dHsaCPE5029120, Bio-Rad) gene labelled with FAM fluorophore (mitochondrial DNA) and the eukaryotic translation initiation factor 2C, 1 (EIF2C1) (assay ID: dHsaCP1000002, Bio-Rad) and ribonuclease P/MRP 30 kDa subunit (RPP30) (assay ID: dHsaCP1000485, Bio-Rad) genes (nuclear DNA) labelled with HEX.

Genomic DNA from edited cells was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and the percent edited alleles measured for an in-out PCR product, amplified with primers IGHG1-HA-Fwd2, IO-EEK-In-6, and probe sg05-P1 (5’ FAM; ZEN/3’-IBFQ double quenched) (Supplementary Table 5). A human RPP30 copy number assay labeled with HEX (Bio-Rad, dHsaCP1000485) was used to measure total allelic copy numbers. Droplets were prepared using ddPCR Supermix for Probes (No dUTP) and a QX200 Droplet Generator (Bio-Rad). The PCR reaction was run on a C1000 Touch Thermal Cycler with the following conditions: 95° C 10 mins (ramp 2°C/sec), 40 cycles [94°C 1 min (ramp 1°C/sec), 55°C 30 sec (ramp 2°C/sec), 72°C 2 min (ramp 1°C/sec)], 98°C 10 min (ramp 2°C/sec), and 4°C forever (ramp 2°C/sec). Droplets were read on a QX200 Droplet Reader and the copy number variation data was analyzed with QuantaSoft analysis software (Bio-Rad).

To determine FGFR2 copy number, we used droplet digital PCR (ddPCR) with a QX200 instrument (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Briefly, gDNA was first digested by EcoRI-HF (NEB) and cleaned up by AMPure XP beads (Beckman Coulter). Digested gDNA (4 ng) was assayed per 20-μl reaction. The copy number assay primers and probes for FGFR2 (dHsaCP2500320) and RPP30 (dHsaCP1000485) reference were obtained from Bio-Rad. After droplet generation, the reaction mixes proceed to thermal cycling as 95 °C × 10 min (1 cycle), 94 °C × 30 s, and 60 °C × 60 s (40 cycles), 98 °C × 10 min (1 cycle), and 12 °C hold. Droplet fluorescence was determined and the QuantaSoft software (Bio-Rad) was used to determine copy number. FGFR2 copy number was estimated as the ratio of the FGFR2 and RPP30 copy number multiplied by two. Each sample was measured in triplicate. As a positive control and standard curve for comparison, we used a gDNA mixture with different ratios of Kato III, a DGC cell line with a known FGFR2 amplification, and a normal DNA source, NA18507 gDNA (Coriell).

Droplet digital PCR assays consisted of the following components (final concentrations in 20 uL total reaction volume): ddPCR SuperMix for Probes (no dUTP) (1x, Bio-Rad), forward primer (900 nM), reverse primer (900 nM), Reference probe (FAM or HEX, 250 nM), NHEJ/drop-off probe (different fluorophore than reference; FAM or HEX, 250 nM), restriction enzyme (AluI, 4 units), nuclease-free water, and gDNA. All primers and probes were designed using Primer3 plus (http://primer3plus.com) and purchased from IDT DNA. All probes included the ZEN internal quencher and 3’ Iowa Black FQ quencher. All ddPCR assays were analyzed using the QX200 droplet reader and Quantasoft software version 1.7.4 (Bio-Rad). Standard ddPCR thermal cycling conditions were used for the SFRP1 assay, with an annealing temperature of 55°C. For NODAL assays, a “3-step” protocol was used, with an annealing temperature of 56°C and an additional 2 minute extension step at 72°C performed for each cycle. For copy number analysis, an RPP30 assay (Bio-Rad; dHsaCP1000485) was used as a reference and SFRP1 FAM assays were performed in parallel with 20 ng of gDNA loaded for each reaction.