Autodg system

ddPCR reaction for Neat1 knock-down control were performed with the Bio-Rad system. The ddPCR reaction mixture (22 μl) contained 2 x QX200 ddPCR EvaGreen Supermix (no dUTP) (Bio-Rad), 2 μM of a mix of forward and reverse primers (Supplementary file 2), and 2/4/6 μL of cDNA depending on the target. The reaction mixture was transferred for droplet generation by AutoDG System (Bio-Rad) in individual wells of disposable DG32 Automated Droplet Generator Cartridges that were already placed in the cartridge holder. The droplet was generated by AutoDG System, between 15000–20000 droplets/well. The prepared droplet emulsions were further loaded in ddPCR 96-Well Plates (Bio-rad) by aspirating 40 μl from the DG32 cartridge by the AutoDG System. The plate was then heat sealed with pierceable foil using a PX1 PCR plate sealer 5 s at 180 °C (Bio-Rad), and PCR amplification was carried out in a T100 thermal cycler (Bio-Rad). The thermal consisted of initial denaturation at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s (denaturation) and 60 °C for 1 minute (annealing/elongation) with a ramp of 2 °C/s, a signal stabilization step at 4 °C 5 min followed by 90 °C 5 min. After PCR amplification the positive droplets were counted with a QX200 droplet reader (Bio-Rad).