Droplet generation oil for probe

Immune cell RNA was quantified using the Qubit fluorometer and RNA BR assay kit (Thermo Fisher), and cDNA was synthesized from ≥10 ng of RNA per sample using qScript cDNA supermix (Quantabio). cDNA was combined with ddPCR supermix and droplet generation oil for probes (Bio-Rad), and droplets were generated using the Bio-Rad QX200 Droplet Generator. PCR was performed using IFNLR1 and GAPDH probes according to the manufacturer's instructions, and droplet fluorescence was analyzed using the Bio-Rad QX200 Droplet Reader. Absolute quantification of transcript number was determined using QuantaSoft Analysis Pro software.

In all the following
experiments, the device configuration was
fixed with 34 Ga needles, 80 μL of oil phase, no additional
oil at the Luer-lock, and 150g centrifugation run
for 5 min. The droplet and Gelbead generation using the described
device was respectively characterized with PCR mix, LAMP mix, and
culture media mix. In each 20 μL of reaction mixture, the PCR
mix contained 1× ddPCR Supermix and 50 μM calcein; the
LAMP mix contained 1 × WarmStart LAMP Mastermix, and 50 μM
calcein; the culture media mix was TSB with 1 mg/mL BSA (New England
Biolabs) and 50 μM calcein. The mix was briefly pipet-mixed.
The reaction mix for Gelbead generation contained 7.5 w/v% PEG hydrogel,
added as 10× PEG monomers. For dispersion of PCR mix as droplets
and Gelbeads, Droplet Generation Oil for Probes (BioRad) was used
instead of fluorinated oil with 5% FluoroSurfactant.
For thermal
stability characterizations, generated droplets or Gelbeads were extracted
into PCR tubes (0.2 mL individual PCR tubes, BioRad) and incubated
in a thermal cycler (T100, BioRad). The thermocycling protocol for
PCR included 10 min of initiation at 95 °C, followed by 40 cycles
of denaturation at 94 °C for 30 s, annealing at 52 °C for
60 s, and extension at 65 °C for 30 s. For LAMP heating, droplets
or Gelbeads were incubated at 65 °C for 1 h.

To determine miR-4649-5p expression by ddPCR, RNA was isolated using TRIzol™ (Thermo Fisher Scientific) as indicated by the manufacturer and 10 ng total RNA were reverse transcribed with the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific) according to the protocol. cDNA was diluted 1:100 and 5 μl were used per 20 μl reaction with 10 μl ddPCR Supermix for Probes (No dUTP; BioRad) and 0.5 μl TaqMan Advanced miRNA Assay for miR-4649-5p (Thermo Fisher Scientific). Droplets were generated from the samples in duplicates with a QX200™ droplet generator (BioRad) using Droplet Generation Oil for Probes (BioRad) and DG8™ Cartridges for QX200™ (BioRad) according to the manufacturer. Droplets were transferred into semi-skirted 96-well ddPCR plates (BioRad), plates were heat-sealed, and PCR was performed on a T100TM Thermal Cycler (BioRad) (95 °C 10 min; 95 °C 30 s + 61 °C 1 min × 39; 98 °C 10 min; ramping 2 °C/s). The droplets were read on a QX200™ droplet reader device (BioRad) and data were evaluated using the QX Manager Software Version 1.2 (BioRad).

The QX200 Droplet Digital PCR System (BioRad) was used to analyze all ddPCR results. ddPCR samples were prepared with 10 μL of ddPCR with ddPCR Supermix for Probes (Bio‐Rad), 1 μL of primer and probe mix (solution of 10 μM target probe and 20 μM reverse/forward primers), 1 μL of template/TE buffer, and 8 μL of water. Once prepared, 20 μL of each reaction and 70 μL of Droplet Generation Oil for Probes (Bio‐Rad) were loaded into DG8 Cartridges and covered with DG8 Gaskets. Using the QX200 Droplet Generator, water−oil emulsion droplets were created. Cycle conditions for PCR was based on optimized conditions from BioRad. For each biological rep, three technical repetitions were completed. Unless stated otherwise, technical reps were averaged. Technical reps were only excluded if saturated was detected or there were inconsistent positive event amplitudes.

RT-ddPCR was performed with the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, Hercules, United States). The reaction mix contained 500 nM forward primer, 900 nM reverse primer and 100 nM probe. Each sample (5.5 µL sample and 16.5 µL reaction mix, i.e. 20 µL + 10%) was applied to a 96-well plate, with a final volume of 22 µL. A 20 µL aliquot was then transferred to DG8™ Cartridges (Bio-Rad), and 70 µL of Droplet Generation Oil for Probes were added (Bio-Rad). Droplets were generated with a QX200™ Droplet Generator (Bio-Rad). After droplet generation, 40 µL of droplet suspension (containing the entire 5 µL of sample and 15 µL of reaction mix) were transferred to a 96-well plate (Eppendorf, Hamburg, Germany). RT-PCR was performed in a T100™ Thermal Cycler (Bio-Rad) with RT at 50 °C for 1 h, reverse transcriptase inactivation and enzyme activation at 95 °C for 10 min, followed by 50 cycles of denaturation at 95 °C for 30 s, annealing/elongation at 60 °C for 1 min and a final enzyme deactivation step at 98 °C for 10 min. Plates were transferred to the QX200™ Droplet Digital™ PCR system (Bio-Rad) on either the same day or the day after the reaction. Results were visualised in QuantaSoft™ software version 1.7.4 (Bio-Rad). Thresholds were set manually using the fluorescence intensity of the no template controls (NTCs) within each run as reference.