Fig. 5

CfRNA peaks are recurrently protected by proteins, vesicles, and local structures. Recurrently protected regions in the reference dataset (GSE71008). A Illustration of (RBP) protected peak region in cell-free environments. B Scaled meta plot of reads coverage and tWPS around peak center. Dash line means peak center position. Ribbon area means 95% CI calculated from multiple regions. C Left: bar plot showed ratio of RBP-, EV-, and RGS-intersected peak number to total in peak and size-matched background regions. Right: bar plot showed MFE and mean mRNA icSHPAE reactivity value in the extended peak and size-matched background regions. Error bar means SEM calculated from multiple samples. D Scaled frequency meta plot and heatmap of annotated RBP-binding sites, EV-sorting sites, and RGS sites occurrence in the extended peak regions. Ribbon area means 95% CI calculated from multiple samples. Regions in heatmap were ranked by mean of scaled occurrence. Representative motif patterns enriched from AGO2-binding-sites-overlapped, EV-sorting, and RGS peak regions were shown below. E Upset plot showed overlapped contribution of four protection factors in all annotation-overlapped consensus peaks. Venn plot was shown in the top right. F Stacked bar plot showed the percentage of top 5 RBPs (ranked by overlapped peak number) by transcript species in all RBP-binding-sites-overlapped consensus peaks. RGS related RBPs (G4RBP) were highlighted in black triangle. *P value < 0.05, **P value < 0.01, ***P value < 0.001, ****P value < 0.0001, Wilcoxon rank sum test, one-tailed (peak has higher ratio and lower MFE or reactivity than background). MFE, minimum free energy; RGS, RNA G4 structure; RSS, RNA secondary structure; tWPS, transcriptomic window protection score. FC: fold change (peak/background). Delta: difference (peak-background)