Coumarin acetate

PCR droplets were reinjected and spaced into a fusion device at a rate of ∼1500 droplets/sec. Each PCR droplet was then synchronized with a 16 pL IVT droplet containing 2 mM each NTP (Larova), 25 mM MgCl₂, 44 mM Tris-HCl pH 8.0 (at 25°C), 5 mM DTT, 1 mM Spermidine, 0.1% of Pluronic F68 (Sigma-Aldrich), 1 µg of pyrophosphatase (Roche), 500 nM Gemini-561, 1 µM coumarin acetate (Sigma-Aldrich) and 17.5 µg/mL T7 RNA polymerase (purified in the laboratory). IVT mixture was loaded in a length of PTFE tubing and kept on ice during all the experiment. PCR droplets were spaced and IVT droplets produced using a dedicated stream of Novec 7500 fluorinated oil (3M) supplemented with 2% (w/w) of fluorosurfactant. Flowrates (MFCS, Fluigent) were adjusted to generate 16 pL IVT droplets and maximize synchronization of 1 PCR droplet with 1 IVT droplet. Pairs of droplets were then fused with an AC field (400 V at 30 kHz) and the resulting emulsion was collected off-chip and incubated for 2 h at 37°C.

DNA mutant libraries were diluted in 200 µg/mL yeast total RNA solution (Ambion) to obtain the desired occupancy of droplets. An amount of 1 µL of this dilution was then introduced in 100 µL of PCR mixture containing 0.5 µM of each primer 3.A (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′) and 1.B (5′-GTACACTGTGCTCGTGTC-3′), 0.2 mM each dNTPs, 20 μM coumarin acetate (Sigma-Aldrich), 0.1% Pluronic F68 (Sigma-Aldrich), 2 U of Q5 DNA polymerase (New England Biolabs) and the corresponding buffer at the recommended concentration. The mixture was loaded in a length of PTFE tubing and infused into a droplet generator microfluidic chip where it was dispersed in 2.5 pL droplets (production rate of ∼12,000 droplets/s) carried by Novec 7500 fluorinated oil (3M) supplemented with 3% of a fluorosurfactant (proprietary synthesis). Droplet production frequency was monitored in real time using an optical device and software developed by the team (Ryckelynck et al. 2015 (link)) and used to determine droplet volume. 2.5 pL droplets were generated by adjusting pumps flowrates (MFCS, Fluigent). The emulsion was collected in 0.2 mL tubes and subjected to an initial denaturation step of 2 min at 98°C followed by 30 cycles of: 10 sec at 98°C, 30 sec at 55°C, 30 sec at 72°C. Droplets were then reinjected into a droplet fusion microfluidic device.