Samples were cycled in a Bio-Rad (Hercules CA, USA) PTC-225 thermal cycler as follows: initial denaturation at 94°C for 2 minutes; 55 cycles at 94°C for 15 seconds, 58°C for 15 seconds and 68°C for 30 seconds; final extension at 68°C for 10 minutes, followed by a 4°C hold. Following PCR amplification, the emulsion of PCR droplets was broken to release each individual amplicon from the PCR droplets. For each sample, an equal volume of RDT 1000 Droplet Destabilizer (RainDance Technologies, 40-00830) was added to the emulsion of PCR droplets, the sample was vortexed for 15 seconds and then centrifuged at 12,000 × g for 10 minutes. The oil from underneath the aqueous phase was carefully removed from the sample. Each sample was purified over a MinElute column (Qiagen, 28004) following the manufacturer's recommended protocol. The sample was eluted off the column with 11 μl of the Qiagen Elution Buffer. Purified amplicon DNA was then analyzed on an Agilent Bioanalyzer to quantify amplicon yield.
