Figure 1.

Method overview. Transcripts are captured on immobilized poly-dT and reverse transcribed. Two oligonucleotide probes are designed against each transcript of interest. The upstream probes contain 20 nt complementary to a universal primer (T7) site, one of 100 different 24 nt barcode sequences, and a 20 nt sequence complementary to the 3'-end of the corresponding first-strand cDNA. The downstream probes are 5'-phosphorylated and contain a 20 nt sequence contiguous with the gene-specific fragment of the upstream probe and a 20 nt universal primer (T3) site. Probes are annealed to their targets, free probes removed, and juxtaposed probes joined by the action of ligase to yield synthetic 104 nt amplification templates. PCR is performed with T3 and 5'-biotinylated T7 primers. Biotinylated barcoded amplicons are hybridized against a pool of 100 sets of optically addressed microspheres each expressing capture probes complementary to one of the barcodes, and incubated with streptavidin-phycoerythrin to label biotin moieties fluorescently. Captured labeled amplicons are quantified and beads decoded by flow cytometry. nt nucleotides.