Each transcript of interest was targeted with an upstream and downstream probe pair. Upstream and downstream probes were each designed with a 20nt gene specific region (40nt contiguous sequence per probe pair), a unique identifying barcode, and a universal primer site. The gene specific sequences were blasted against the human genome to verify that each is unique to the targeted gene of interest, as described in the steps below. In addition to gene specific sequence, upstream probes contained a T7 primer site, and a 24-nucleotide (nt) barcode, and downstream probes, which were 5′ phosphorylated, contained the T3 primer site. Barcode sequences are shown in supplementary Table S2. Probes were synthesized by IDT (Integrated DNA Technologies).
