Extended Data Fig. 3. In vitro SHAPE reactivity of the RORC RNA switch sequence in vitro.

a SHAPE reactivity profiles for the reference sequence and for the mutation–rescue pair of sequences (blue - ‘77-GA’, red - ‘63-TC,77-GA’). Shown is the average for 3 replicates with the respective error bars (SD). The SHAPE reactivity changes in the nonmutated regions are highlighted in bold arrows. b Barplots of cumulative SHAPE reactivity within the switching regions for the reference sequence (in gray) and for the mutation–rescue pair of sequences (blue - ‘77-GA’, red - ‘63-TC,77-GA). N replicates = 3. c Scatter plot showing the reproducibility of the DMS signal between two replicates. Each dot represents a single nucleotide. Normalized DMS signal is shown on both axes. Correlation and P value is determined with Pearson correlation coefficient (P = 1.59-42). d Scatter plots showing the reproducibility of the DRACO clusters between replicates (N = 2). Each replicate’s reads were clustered with DRACO, the DMS reactivity was calculated for each cluster; the clusters were subsequently matched between replicates. Shown are DMS reactivities for a given cluster in a given replicate; each dot represents a single nucleotide. Correlation and P value is determined with Pearson correlation coefficient. P values left to right: 2.60e-23,3.62e-07,0.18,0.73. e DMS reactivities of the two clusters identified by the DRACO unsupervised deconvolution algorithm (Morandi et al. ²⁸). The algorithm was run on two replicates independently, and identified the same clusters in both of them. The ratios of the clusters reported by DRACO are 22% to 78% in replicate 1 and 32% to 68% in replicate 2. The ratio shown is an average between the two replicates. The switching regions are shown in color. f The effect of sequence mutations in the ‘Box 2’ and ‘Box 3’ regions of RORC element on their reactivity, as measured by DMS-MaPseq in a reporter cell line. P values were determined using the two-sided independent T-test. g Correlation of relative proportions of the two conformations between the reporter context and the endogenous RORC mRNA. Linear regression is shown with a line. The relative conformations’ proportion is defined as the ratio of reactivities of Box 2:Box 3.