Quantitative Droplet Digital PCR Protocol

Droplet digital PCR (ddPCR) was carried out according to manufacturer recommendations and as described^{43 (link), 44 (link)}. Briefly, the ddPCR mixture contained 7.5 μl of 2× ddPCR Evagreen Supermix (Bio-Rad, Hercules, USA), 10 nM of both the Zeb2-NAT, Zeb2 intron 1, or GAPDH forward and reverse primers, and 5 ng of cDNA (RNA equivalent) in each 15-μl reaction. The 15-μl reaction was assembled into a droplet cartridge (Bio-Rad, Hercules, USA), according to the Bio-Rad protocol and the cartridge was placed in the droplet generator (Bio-Rad #186-3002) giving rise to around 20,000 individual droplets in an emulsion^{43 (link)}. Droplets were transferred to a 96-well plate (Eppendorf, Hamburg, Germany) and thermal sealed with a foil lid (Eppendorf, Hamburg, Germany). Sealed plates were cycled using a Veriti DX thermal cycler (ThermoFisher Scientific) under the following conditions: 2 min at 30 °C; 10-min hold at 95 °C; 48 cycles of 95 °C for 50 s and then 59 °C for 120 s; 5 min at 4 °C; and 5 min at 90 °C. After amplification, the plate was transferred to a Bio-Rad droplet reader (QX200) from which data were extracted and analyzed with the Quantasoft software following manufacturer recommendations.