2h perfluoro 1 octanol

The emulsion was finally reinjected into an analysis and sorting microfluidic device at a frequency of ∼150 droplets/sec and spaced with a stream of surfactant-free Novec 7500 fluorinated oil (3M). The orange fluorescence (Gemini-561 in complex with an aptamer) of each droplet was analyzed and the most orange fluorescent droplets were sorted (from 6.7% to 0.18% depending on the round of µIVC, Supplemental Table S1). The gated droplets were deflected into collecting channel by applying an AC fields (1000 V at 30 kHz) and collected into a 1.5 mL tube. Sorted droplets were recovered from the collection tubing by flushing 200 µL of HFE fluorinated oil (3M). An amount of 100 µL of 1H, 1H, 2H, 2H-perfluoro-1-octanol (Sigma-Aldrich) and 200 µL of 200 µg/mL yeast total RNA solution (Ambion) were then added and the droplets broken by vortexing the mixture. DNA-containing aqueous phase was then transferred into a classical Eppendorf tube.

The emulsion was finally reinjected into of analysis and sorting microfluidic device mounted on Thermo plate (Tokai Hit) holding the temperature at 45°C. Prior to starting droplet fluorescence analysis and sort, the temperature of the microfluidic device was allowed to equilibrate at 45°C for 10 min. The proper warming of the chip was controlled with a thermocouple. The micrometric depth of the channels together with the low droplets re-injection flow rate used were expected to allow equilibration of droplets temperature to that of the device prior to analyzing their fluorescence. Droplets were reinjected at a frequency of ≈200 droplets/s and spaced with a stream of surfactant-free HFE 7500 fluorinated oil (3M). The green fluorescence (DFHBI complexed with the aptamer) of each droplet was analyzed and the 1–2% most green fluorescence droplets were sorted. The gated droplets were deflected into collecting channel by applying a 1 ms AC fields (1200 V 30 kHz) and collected into a 1.5 ml tube. Residual droplets were recovered from the collection tubing by flushing 200 μl of HFE fluorinated oil (3M). 100 μl of 1H, 1H, 2H, 2H-perfluoro-1-octanol (Sigma-Aldrich) and 200 μl of 200 μg/ml yeast total RNA solution (Ambion) were then added and droplets broken by vortexing the mixture. DNA-containing aqueous phase was then recovered.