Assay loading reagent 2x

Real-time PCR was performed using Fluidigm 96.96 Dynamic Arrays with BioMark™ HD system (Fluidigm, South San Francisco, CA, USA), according to the manufacturer's protocol. Briefly, 2.25 μL cDNA was combined with 2.5 μL TaqMan Universal PCR Master Mix (2X) (Thermo Fisher Scientific) and 20X GE sample loading reagent (Fluidigm), before loading onto a 96.96 Dynamic Array integrated fluidic circuit. Similarly, 2.5 μL TaqMan assays (Table S2) and assay loading reagent 2X (Fluidigm) were combined before loading onto a 96.96 Dynamic Array integrated fluidic circuit.

3 µL of SNPType ASP1/ASP2 (allele specific primer 1 and 2) and 8 µL of SNPtype LSP were premixed and dilute with 29 µL of DNA suspension buffer to prepare SNPType assay mix. Each assay (5 µL) comprised 1 µL SNPType assay mix, 2.5µL assay loading reagent 2X (fluidigm) and 1.5 µL DNA-free water. Each sample (6 µL) comprised 3 µL Biotium fast probe master mix 2X (Biotium), 0.3 µL sample loading reagent 20X (fluidigm), 0.1 µL SNPType reagent 60X (Fluidigm), 0.036 µL ROX 50X (Invitrogen), 0.064 µL DNA-free water and 2.5µL amplified genomic DNA. Each of the assays (4 µL) and samples (5 µL) was pipetted into separate inlets in the chip. Amplification was carried out under the following conditions: 95°C for 5 min, 38 cycles of 95°C for 15 s, 60°C for 45 s, 72°C for 15 s followed by 30 s at 20°C for fluorescence measurement.

Each assay (5 µL) comprised 2.5 µL assay loading reagent 2X (fluidigm), 0.25 µL ROX 50X (Invitrogen), 1.25 µL SNP Genotyping assay mix 40X (Applied Biosystems) and 1 µL DNA-free water. Each sample (6 µL) comprised 3 µL TaqMan universal PCR master mix 2X (Applied Biosystems), 0.3 µL sample loading reagent 20X (fluidigm), 0.3 U AmpliTaq gold polymerase (Applied Biosystems), 0.12 µL DNA-free water and 2.52 µL amplified genomic DNA. Each of the assays (4 µL) and samples (5 µL) was pipetted into separate inlets in the chip. Amplification was carried out under the following conditions: 50°C for 2 min, 98°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 60 s. Fluorescence was measured at each slice.