Ddpcr droplet reader oil

In order to specifically enrich fluorescent droplets, sorting was performed with a set-up as previously described (Chaipan et al., 2017 (link); Debs et al., 2012 (link)). Droplets were reinjected into the sorting device at a frequency of ca. 100 Hz–250 Hz (flow rates 15–30 μL/h) using syringe pumps (Harvard apparatus) along with two spacer sheath oil inlets (ddPCR™ Droplet Reader Oil, Bio-RAD) at twenty times higher flow rates. To detect the fluorescent 6-FAM signal coming from cleaved TaqMan probes during the PCR reaction, droplets were excited using a blue diode laser (488 nm, 20 mW; Melles Griot) and the fluorescence intensity was measured at and upwards of the emission wavelength of 514 nm using a PMT (Hamamatsu). A custom LabVIEW software was used to enable dynamic adjustments of PMT gain (0–1 V), droplet fluorescence width and intensity thresholds for sorting, electrode voltage (1–1.5 kV), AC pulse frequency (30–40 kHz), pulse duration and delay.

Droplet Generation Oil, ddPCR Droplet Reader Oil, and 2X ddPCR SuperMix for Probes (no dUTP) were purchased from Bio-Rad. Primers and probes (see below) were purchased from Integrative DNA Technologies as was gBlock reference DNA. The latter was double-stranded DNA corresponding to the predicted amplicon sequences and was stored at concentrations of 1e9 copies/μL in 1:1 Tris (10 mM)-EDTA (1 mM) (pH 7.8)/glycerol with 1 μg/mL sonicated salmon sperm gDNA (Abnova). The transfer plasmid used in manufacturing AVGN7 was also used as a reference standard and was generated by GenScript. All ddPCR assays were performed using the Bio-Rad system. This includes the QX200 Droplet Generator, the QX200 Droplet Reader, the C1000 Touch PCR Thermal Cycler, the PX1 Plate Sealer, and various consumables (e.g., plates, heat seal foil, droplet cartridges) designed to work with the system. The Quantstudio 7 Pro qPCR System (Applied Biosystems) was also used in the initial assessment of primer/probe sets.