Single-Cell Gene Expression Analysis

For cDNA synthesis and preamplification of target genes CellsDirect One-Step qRT-PCR kit (Invitrogen) was used. Cells were sorted directly into PCR plates or 0.2 mL PCR tubes containing 2.5μL gene specific 0.2x TaqMan gene expression assays (Applied Biosystems), 5μL of CellsDirect 2x Reaction mix (Invitrogen), 1.2μL CellsDirect RT/Taq mix, 1.2μL TE buffer and 0.1μL SUPERase-In RNase Inhibitor (Ambion) to a total volume of 10μL. Conditions for reverse transcription and target gene amplification were: 15 min at 50°C; 2min at 95°C; 22 cycles of 95°C for 15s and 60°C for 4min. Preamplified products were diluted 1:5 in TE buffer and analyzed on a 48x48 Dynamic Array (Fluidigm) using the following PCR cycling condition: 95°C for 10min; 40 cycles of 95°C for 15s and 60°C for 60s. Data was analyzed using the ΔΔCt method as described44 (link); results were normalized to B2m. Single cells not expressing Hprt or Actinb or B2m were excluded from the analysis. Taqman assays used for multiplexed quantitative real-time PCR are listed in Supplementary Table 4.