Ddpcr supermix for probes no udp

One RMS cell per well was sorted in 96-well plates containing 4.5 μl Single-Cell Lysis Buffer and 0.5 μl Single-Cell DNase I (Single-Cell Lysis Kit, 4458235; Ambion) using a Moflo Astrios. The reaction was stopped by adding 0.5 μl of single-cell stop solution. cDNA synthesis was performed after adding 2.5 μl of RT reaction mix (iScript Advanced cDNA Synthesis kit, 1725038; Bio-Rad) for a total volume of 10 μl. Droplet digital PCR amplification of Pax3:Foxo1 (FAM) and Gapdh (HEX) was performed in a final volume of 25 μl by adding the ddPCR Supermix for Probes No UDP (Bio-Rad), the FAM and HEX probes to the cDNA mix. Droplets were generated using the QX100 Droplet Generator (Bio-Rad) with 70 μl Droplet Generation Oil (Bio-Rad) and 40 μl of the resulting water-in-oil droplet emulsion was then thermocycled at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 61.3°C for 1 min. Final enzyme deactivation took place at 98° for 10 min. Individual droplets were analyzed using the Q100 Droplet Reader and Quantasoft Software. The following probe sequences were used: 5′-/56-FAM/CATTGGCAA/ZEN/TGGCCTCTCACCTCAGAA/3IABkFQ/-3′ (P3F FAM probe), 5′-/5HEX/ACCACAGTC/ZEN/CATGCCATCACTGCCACC/3IABkFQ/-3′ (GAPDH HEX probe).