Primed 96.96 dynamic array chip

Total RNA was purified using the RNeasy Plus Micro Kit (Qiagen). cDNA was synthesized using all RNA available (or 1–5 ng) with the High-Capacity Reverse Transcription Kit with RNase Inhibitor (Life Technologies) (25° C for 10 min, 37° C for 120 min, 85° C for 5 min). cDNA equivalent to 1000 sorted cells was subjected to gene-specific preamplification using Taqman Preamp MasterMix (Applied Biosystems) and 96 pooled TaqMan Assays (Applied Biosystems) (Supplementary Tables 21–23) at final concentration 0.2X (95°C for 10 min, followed by 16 cycles of 95°C for 15 s and 60°C for 4 min). The preamplified cDNA was diluted 5-fold in DNA suspension buffer (Teknova) and was mixed with TaqMan Universal PCR Master mix (Life Technologies) and 20X GE sample loading reagent (Fluidigm). 20X Taqman assays were diluted 1:1 with 2X assay loading buffer (Fluidigm). Taqman assays mixtures were loaded onto a primed 96.96 Dynamic Array chip (Fluidigm). The chip was loaded into the IFC Controller, where each sample was mixed with each assay in every possible combination. The chip was transferred in a Biomark (Fluidigm) for real-time PCR amplification and fluorescence acquisition using single probe (FAM-MGB, reference: ROX) settings and the default hot-start protocol with 40 cycles. Cycle thresholds (Ct) were calculated using the Fluidigm Biomark software v1.4.2.