Biorad qx200 automated droplet generator

Prior to ddPCR, various sample types were diluted to different degrees in nuclease-free water: sorted-PGC lysates (4×), dissociated-embryo lysates (3000–6000×), whole-embryo lysates (10×), and whole-adult lysates (30 adults lysed in 60 µL lysis buffer, 1000×). The ddPCR was run according to the manufacturer’s recommendations. Briefly, ddPCR reactions were assembled as 24 µL mixes containing 0.1 µM of each primer, Bio-Rad QX200 ddPCR EvaGreen Supermix (BioRad 186–4034), 0.1 U/µL SacI-HF (New England Biolabs), and 4.8 µL of the sample. Reactions were incubated in the dark at room temperature for 30–60 min to allow SacI-HF (NEB R3156) digestion to linearize/digest DNA prior to droplet generation. After incubation, samples were loaded for droplet generation in a BioRad QX200 Automated Droplet Generator. The PCR amplification was performed as follows: 10 min at 95°C, 40 cycles of 94°C for 30 s, and 60°C for 1 min, followed by 10 min at 98°C for all primer pairs. Samples were all run in triplicate and were immediately analyzed using a BioRad QX200 Droplet reader. All ddPCR reactions were single oligo-pair mixes; therefore, absolute DNA concentrations were calculated using 1D-amplitude plots in BioRad QuantaSoft software.

Prior to ddPCR, various sample types were diluted to different degrees in nuclease-free water: sorted-PGC lysates (4×), dissociated-embryo lysates (3000–6000×), whole-embryo lysates (10×), and whole-adult lysates (30 adults lysed in 60 µL lysis buffer, 1000×). The ddPCR was run according to the manufacturer’s recommendations. Briefly, ddPCR reactions were assembled as 24 µL mixes containing 0.1 µM of each primer, Bio-Rad QX200 ddPCR EvaGreen Supermix (BioRad 186–4034), 0.1 U/µL SacI-HF (New England Biolabs), and 4.8 µL of the sample. Reactions were incubated in the dark at room temperature for 30–60 min to allow SacI-HF (NEB R3156) digestion to linearize/digest DNA prior to droplet generation. After incubation, samples were loaded for droplet generation in a BioRad QX200 Automated Droplet Generator. The PCR amplification was performed as follows: 10 min at 95°C, 40 cycles of 94°C for 30 s, and 60°C for 1 min, followed by 10 min at 98°C for all primer pairs. Samples were all run in triplicate and were immediately analyzed using a BioRad QX200 Droplet reader. All ddPCR reactions were single oligo-pair mixes; therefore, absolute DNA concentrations were calculated using 1D-amplitude plots in BioRad QuantaSoft software.

DNA was pre‐amplified using primers flanking the codon 12 region (see Supplementary materials and methods). Droplet digital PCR (ddPCR), with a multiplex ddPCR screening assay to pre‐screen samples, was used to detect six KRAS codon 12 variants (c.34G>T (p.G12C), c.35G>A (p.G12D), c.34G>C (p.G12R), c.35G>T (p.G12V), c.35G>C (p.G12A), c.34G>A (p.G12S)). Empirical testing showed the combination of c.34G>T (p.G12C), c.35G>A (p.G12D), and c.34G>C (p.G12R) probes (Thermo Fisher Scientific) was sufficient to detect and resolve unique clusters for all KRAS variants (supplementary material, Figure S2). Any positive variant was confirmed with a variant‐specific ddPCR assay that was also used to establish variant allele frequency (Supplementary materials and methods; and supplementary material, Table S2). Note that enrichment of the endometriotic epithelium was not done (for LCM or macrodissection) and thus the variant allele frequency of the epithelial fraction is not known. Droplets were generated using a BioRad QX200 Automated Droplet Generator (BioRad Laboratories, Hercules, CA, USA), and quantified on a BioRad QX200 Droplet Reader (BioRad Laboratories). The mean detection thresholds and ranges for the individual KRAS assays in this study are listed in supplementary material, Table S3.