Figure 2.

Optimization of library preparation and anchor primer hybridization on-chip. Optimization experiments, showing the RCP yield for (a) padlock probing, (b) RCA, and RCPs’ mean SNR for (c) anchor primer hybridization, all normalized to off-chip performance. Standard and optimized parameters correspond to the outermost left and right bars, respectively in (a–c). Incubation time parameter formulated as α × β′+ ʎ′ reads as α deliveries incubated β min each, followed by ʎ min final incubation. (d) Performance obtained by combining the optimized parameters derived from (a–c) on-chip, normalized to the manually performed control off-chip. For (a–c) all bars n = 2, biological replicates. For (d) n = 3 technical replicates. The red squares highlight the first occurence of a parameter’s final value, which was retained in the optimized, device-enabled, protocol.