detector inlet. The chip was then placed in the NanoFlexTM 4-IFC Controller (Fluidigm) for loading and mixing. After loading, the chip was processed in the BioMarkTM Real-Time PCR System (Fluidigm) using a cycling program of 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s and 72 °C for 30 s. After completion of qPCR, a melting curve of amplified products was determined. Data were collected using BioMarkTM Data Collection Software 2.1.1 build 20090519.0926 (Fluidigm) as the cycle of quantification (Cq), where the fluorescence signal of amplified DNA intersected with background noise. Data preprocessing and analysis was completed using Fluidigm Melting Curve Analysis Software 1.1.0 build 20100514.1234 (Fluidigm) and Real-time PCR Analysis Software 2.1.1 build 20090521.1135 (Fluidigm) to determine valid PCR reactions. Invalid reactions were removed from later analysis. Data was analyzed following published protocols for single cell RT-qPCR40.
