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Page 10 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

fully secure. Run the DG8 droplet generator according to the manufacturer’s instructions.

Following droplet generation, aspirate 40 µL of the droplets by slowly withdrawing the pipette from the
bottom of the well at a 35-degree angle over 5 s. Note: handle the droplets with extreme care to avoid
damaging them and do not freeze. Transfer the droplets into a deep-well 96-well plate (Bio-Rad catalog
number: 12001925) by touching the pipette tip to the side of the well, about halfway down. Transfer droplets
into the well slowly for 5 s, taking care not to introduce bubbles.

Seal the 96-well plate using the pierceable foil and Bio-Rad PX1 PCR Plate Sealer according to the
manufacturer’s instructions. In short, place a foil seal (Bio-Rad Catalog number: 1814040) with the red line
facing away from the wells of the plate, ensuring that only one seal is used. Ensure that the plate sealer block
is at room temperature, then place the 96-well plate on the plate sealer block, and cover it with the plate
sealer frame and apply pressure at 180 °C for 5 s. Check that the foil is properly sealed by looking for circles
etched in the foil where the well openings are located.

Immediately following the plate sealing process, transfer the deep well 96-well plate into the thermocycler
set to the following conditions: 95 °C for 10 min (ramp 2 °C/s); 94 °C for 30 s (ramp 2 °C/s); 60 °C for 1 min
(ramp 2 °C/s); repeat at step 2, 39 times; 98 °C for 10 min (ramp 2 °C/s); hold at 4 °C indefinitely; lid
temperature: 105 °C. Following PCR, the droplets may be stored at 4 °C for no more than 24 h. Do not
freeze and proceed to droplet reading and data analysis.

7. Droplet reading and data analysis [Figure 2F]:

After PCR is performed on the droplets, let the 96-well plate rest at room temperature for 5 min prior to
transferring to the QX200 Droplet Reader. Follow the instructions for the Bio-Rad QX200 Droplet Reader
using the official Bio-Rad QX200 Droplet Reader Instruction Manual. The manual provides detailed
guidance on the setup, operation, and maintenance of the instrument, including step-by-step procedures,
safety considerations, and troubleshooting tips. It is essential to read and follow the instructions carefully to
ensure best practices and obtain accurate results. For specific questions or further assistance, contact Bio-
Rad directly or consult their technical support resources.

Follow the instructions in the Bio-Rad QuantaSoft Software Instruction Manual for data analysis using Bio-
Rad QuantaSoft Software. The manual is a comprehensive guide and provides step-by-step instructions for
data analysis, including data import, setting analysis parameters, generating reports, and interpreting
results. Follow the instructions provided in the manual carefully to ensure accurate and reliable data
analysis. If you have specific questions or need further assistance, contact Bio-Rad directly or consult their
technical support resources for expert guidance.

Gating in ddPCR is important for accurate and reproducible data analysis, as it allows the discrimination of
positive from negative droplets, quantification of target molecules, removal of false positives/negatives, and
data normalization. Setting appropriate thresholds ensures reliable and precise results and minimizes
background noise and technical artifacts in ddPCR experiments.

Gating ddPCR results requires several steps. First, use the fluorescence channels to detect the target of
interest (FAM channel) and the spike-in, reference control marker, miR-39 (VIC channel). Next, establish
gating thresholds based on the fluorescence signals to distinguish positive and negative droplets. This can be
done manually or with the automated algorithms provided by the analysis software. Apply the gating