Droplet cartridge

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.

All ddPCR assays used in this study were designed and optimized to work in the ddPCR system by Bio-Rad (Hercules, CA, USA). The Bio-Rad assays IDs are summarized in Supplementary Table 24. The ddPCR mixture contained 8 μl of 2 × ddPCR Supermix (Bio-Rad), 400 nM of forward and reverse primers, 125 nM mutant and wild-tube probe and 2 μl DNA isolated from primary tumour, AAH lesion, plasma or sputum in each 20 μl reaction. The entire 20 μl reaction was loaded into a droplet cartridge (Bio-Rad), a gasket placed over the cartridge according to the Bio-Rad protocol and the cartridge placed in the droplet generator (Bio-Rad #186-3002). Once inside the droplet generator a vacuum was applied to the cartridge. This draws both the PCR reagents and oil through a flow-focusing nozzle where around 20,000 individual droplets ∼1 nl in size are formed, suspended in an emulsion. The emulsion was transferred into a 96 well plate (Eppendorf, Hamburg, Germany) and sealed using a foil lid and a thermal plate sealer (Bio-Rad). Sealed plates were cycled using a C-1000 thermal cycler (Bio-Rad) under the following conditions: 10 min hold at 95 °C, 45 cycles of 95 °C for 15 s then 60 °C for 60 s. After amplification, the plate was transferred to a Bio-Rad droplet reader from which raw fluorescence amplitude data was extracted from the Quantasoft software for downstream analysis.