Quantitative PCR of Single-Cell RNA

PMA and ionomycin in vitro-stimulated CD4⁺ CD25^high T cells were single-cell sorted directly into RT-PreAmp Master Mix (Life Technologies). Cell lysis, sequence-specific reverse-transcription and sequence-specific amplification of cDNA were done as previously described^{37 (link)} and high-throughput quantitative PCR was done on 96.96 Dynamic Arrays with a BioMark system (Fluidigm San Francisco, CA, USA). Amplification curves were quality filtered-using a threshold of 0.65, and melting curves generated for each reaction were manually inspected as an added quality control step. Cycling threshold (C_T) values were calculated with BioMark system software for each assay and the same thresholds were used across all experiments. After exclusion of failed reactions, cells in which the expression of at least three different housekeeping genes mRNA was detected were retained for further analysis. All primers (Fluidigm DELTA gene assays) were pre-validate using positive and negative single cells (data not shown). ΔCt values were calculated in reference to the housekeeping genes.