Evagreen binding dye

The pre-amplification solution in 96 wells included 5 μL of a master mix containing 2.5 μL CellsDirect reaction mix (Invitrogen), 0.5 μL of the primer pool (0.1 μM, Table S1, synthesized at Bioneer), 0.1 μL reverse transcriptase (RT)/Taq polymerase (Invitrogen), and 1.9 μL nuclease-free water. Lysed cells were treated with this mix at 50°C for 1 h, followed by inactivation of RT, activation of Taq at 95°C for 3 min, and 20 cycles of sequence-specific cDNA amplification (15 sec denaturation at 95°C, 15 min annealing and elongation at 60°C). Amplified single-cell cDNAs were first tested in control qRT-PCR reactions for Actb and samples giving Ct values between 13 and 17 were selected for subsequent analysis with the full primer pools, Universal PCR Master Mix (Applied Biosystems) and EvaGreen Binding Dye (Biotium), using 96 X 96 Dynamic Arrays on the BioMark System (Fluidigm). Table S2 lists the Ct values for each gene in each cell, calculated using BioMark Real-Time PCR Analysis software (Fluidigm).

Individual primer sets (total of 96) were pooled to a final concentration of 0.1μM for each primer. Individual cells were sorted directly into 96 well PCR plates loaded with 5μL RT-PCR master mix (2.5μL CellsDirect reaction mix, Invitrogen; 0.5μL primer pool; 0.1μL RT/Taq enzyme, Invitrogen; 1.9μL nuclease free water) in each well. Sorted plates were immediately frozen on dry ice. After brief centrifugation at 4°C, the plates were placed immediately on PCR machine. Cell lyses and sequence-specific reverse transcription were performed at 50°C for 60 minutes. Then, reverse transcriptase inactivation and Taq polymerase activation was achieved by heating to 95°C for 3 min. Subsequently, in the same tube, cDNA was subjected to 20 cycles of sequence-specific amplification by denaturing at 95°C for 15 sec, annealing and elongation at 60°C for 15 min. After preamplification, PCR plates were stored at -80°C to avoid evaporation. Pre-amplified products were diluted 5-fold prior to analysis. Amplified single cell samples were analyzed with Universal PCR Master Mix (Applied Biosystems), EvaGreen Binding Dye (Biotium) and individual qPCR primers using 96.96 Dynamic Arrays on a BioMark System (Fluidigm). Ct values were calculated using the BioMark Real-Time PCR Analysis software (Fluidigm).