Fig. 1: AlkAnilineSeq detects background signals at purine sites in RNA.
From: Mapping of HOCl-oxidized RNA identifies abasic sites as major damage and oxidation product of oxo8G
a Workflow of the AlkAnilineSeq (AAS) protocol. The left panel (1.) details the initial chemical treatment. This process involves fragmentation of RNA under alkaline conditions using a bicarbonate buffer (pH 9.2) at high temperatures (96 °C). The alkaline treatment also renders it more prone to cleavage of certain modified residues. Following fragmentation, existing 5’- and 3’-phosphates, as well as 2’,3’-cyclophosphates in the RNA undergo enzymatic dephosphorylation. Subsequent cleavage with aniline targets the fragile N-glycosidic bond, leading to the formation of new RNA fragments with 5’-phosphates. The right panel (2.) visualizes NGS library preparation and data analysis. During library preparation, adapters ligate to the RNA fragments with 5’-phosphates, facilitating the positive selection of these newly generated fragments. These libraries are then PCR-amplified and sequenced. The resulting read fragments are aligned against a reference RNA with a -1 backshift introduced to align the signal to the modified nucleotide. The AAS signals are indicated by the Stop ratio, derived from the proportion of reads starting at a given position relative to all reads overlapping it. b Background AAS profiles of yeast 25S rRNA ex vivo (naked RNA, n = 3) compared to IVT yeast 25S rRNA (n = 2). RNA positions are indicated on the x-axis, and the Stop ratio is depicted on the y-axis. c Fraction of A, C, G, and U/T for yeast 25S rRNA (ex vivo). d The box-violin plot represents AAS signals at G residues across different rRNA species (5.8S, 18S, and 25S) comparing RNA isolated from yeast (ex vivo, left, n = 3) and IVT rRNA (IVT, right, n = 2). Statistical comparisons were conducted using the Wilcoxon test to evaluate differences across conditions (p-value = 2.40 × 10-40). Box plots show the median (centre line), 25th and 75th percentiles (bounds of box), and whiskers indicate the data points within 1.5x interquartile range (IQR). Source data are provided as a Source Data file. Created in BioRender. Raorane, K. (2025) https://BioRender.com/v1o2mt6.
