Extended Data Fig. 1: RNA release and capture.
a, The workflow to identify the optimal release temperature. Gel with anchored transcripts was heated for 30 min at different temperatures to release RNA molecules, which were collected and quantified on a Bioanalyzer. b, Representative bioanalyzer gel image from three replicates, showing RNA released from the gel at different incubation temperatures. c, Representative bioanalyzer traces showing the released RNA. Only weak rRNA peaks are expected as the RNA anchoring is mediated by polyA. d, Released RNA concentrations at different incubation temperatures measured from three biological replicates. Error bars: mean ± standard derivation (SD). Increasing temperature from 39 °C to 41 °C significantly increased RNA concentrations, whereas no significant difference was observed above 41 °C. P values: two-sided Welch’s t-test. e, Violin plot showing the number of UMIs per gene detected by Ex-ST and Visium using standard and modified protocols. f-g, Comparisons of number of genes (f) and UMIs (g) per tissue area (20 by 20 µm) measured by Ex-ST and Visium using the standard and modified protocols, calculated by normalizing the number of genes and UMIs per spot by the tissue area covering each spot. Ex-ST and standard Visium data in e-g were downsampled to match sequencing saturation (Extended Data Table 1). P values: two-sided, two-sample Mann-Whitney-Wilcoxon tests.
