Fig. 1
From: Protocol optimization improves the performance of multiplexed RNA imaging
The Role of Encoding Probe Design and Hybridization on Single-Molecule Signal Brightness. (A) Images of U-2 OS cells stained with encoding probes that contain 20-nt (left), 30-nt (middle), or 50-nt (right) target regions and which were stained with optimal formamide (FA) concentrations for those lengths (10%, 30%, and 50%, respectively). Two different mRNAs were targeted: SCD (top) or CSPG4 (bottom). (B, C) Single-molecule signal brightness for encoding probes with different target region lengths and stained with different formamide concentrations (bar colors) for probes targeting SCD (B) or CSPG4 (C). (D) Images of U-2 OS cells stained with encoding probes targeting 130 RNAs and the first Cy5-labeled readout sequence. Encoding probes were stained at different per-probe concentrations (rows) and for different durations (columns). (E, F) Single-molecule signal brightness for the first Cy5-labeled readout probe (E) and the first AF750-labeled readout probe (F) for samples prepared as in (D) for the listed per-probe concentrations and hybridization durations. (G) Images of U-2 OS cells stained for encoding probes targeting 130 RNAs and the first Cy5-labeled readout sequence when annealed at 50, 60, 70, or 80 °C. A control without this melting and annealing protocol (No Annealing) is also shown. (H) Single-molecule signal brightness for the first Cy5-labeled readout probe (left) or the first AF750-labeled readout probe (right) for samples prepared as in (G) for the listed melting temperature. (I) The fold increase in the average molecular brightness for samples with a 60 °C melting and annealing step and 1 day of hybridization or for samples without a melting and annealing step and 7 days of hybridization as compared to a sample without melting and annealing and 1 day of hybridization. Samples were stained with an encoding probe library targeting 130 RNAs and the first readout probes conjugated to Cy5 or AF750. For A, D, and G: Gray: mRNA signal. Blue: DAPI. Scale bars: 20 μm. For B, C, E, F, H, I: Bars and error bars represent the average or standard deviation across at least two replicates of the average plotted quantities seen within each replicate.
