Fig. 6: The presence of very rare D modifications in human mRNA.
From: Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution
a Strategic illustration of how in vitro transcribed (IVT) mRNA excludes false positives in D detection by CRACI. b Misincorporation signatures revealed by CRACI across 18S rRNA, confirming the absence of false-positive D sites in long RNAs. c CRACI analysis pipeline for D detection in HepG2 mRNA. Uridine sites displaying adequate reads coverage in both cellular mRNA and IVT mRNA are considered. We require the misincorporation ratio in IVT mRNA to be less than 0.2 and in cellular mRNA to be greater than 0.2. Differential misincorporation ratios are analyzed, with a log2 fold change >1 and a p-value < 0.05 (obtained by two-sided t-test) as cutoffs. Additionally, sites must be in mRNA (not tRNA) and undergo multiple cut-off filtering and binomial testing. d Heatmap illustrating D site identified in HepG2 mRNA by CRACI.
