LEAD‐m⁶A‐seq for Locus‐Specific Detection of N⁶‐Methyladenosine and Quantification of Differential Methylation

Abstract

N6‐methyladenosine (m6A) is a crucial RNA chemical mark which plays important roles in various biological processes. The development of highly multiplexed, cost‐effective, and easy‐to‐operate methodologies for locus‐specific analysis of m6A is critical for advancing our understanding of the roles of this modification. Herein, we report a method which builds upon the principle of the previously reported SELECT approach by significantly improving its efficiency and coupling it to next generation sequencing technology for high‐throughput validation and detection of m6A modification at selected sites (LEAD‐m6A‐seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m6A modification at these sites, and estimated differential methylation levels (0–84 %) upon in vitro demethylation by the m6A demethylase FTO with high reproducibility. We envision that this strategy can be readily used for testing a greater number of sites with a broad dynamic range and modified to study other RNA modifications.

Targeting SLC3A2 subunit of system XC− is essential for m6A reader YTHDC2 to be an endogenous ferroptosis inducer in lung adenocarcinoma

“…This cannot exclude that the SLC3A2 mRNA is truly not to be m 6 A methylated, because at some occasions, failure to identify m 6 A methylation only indicates that the abundance of certain modi cation is not high enough to reach the detection threshold [51]. However, such negative results at least manifest that the direct m 6 A regulation of SLC3A2 is less critical in LUAD cells.…”

DENA: training an authentic neural network model using Nanopore sequencing data of Arabidopsis transcripts for detection and quantification ofN6-methyladenosine on RNA

“…As shown in Figure 4h, one synthetic DNA probe with PCR adapter (named Dp) and another synthetic DNA probe (named Up) complementarily anneal to RNA but leave a gap of three nucleotides opposite to the candidate site to be identified. As described in other work[26], most mammalian m 6 A sites are within the G(A/m 6 A)CU motif. To minimize the over extension of the Up probe by Bst 2.0 DNA polymerase, only dATP, dTTP, dGTP, but not dCTP are added to prevent extension across guanosine in G(A/m 6 A)CH (H=A, C or U) [26].…”

“…S3d). In addition, we further compared the predicted modification rates from DENA with those from other methods containing xPore, LEAD-m 6 A-seq and Deoxyribozyme-based Method , at the identified m 6 A sites in previous work[12, 26, 27] (Additional file 1: Table S3). Importantly, we can observe that the modification rates predicted by DENA are consistent with those identified by experimental methods at most sites.…”

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…Native DNA polymerases with RT activity (from Thermus thermophilus in the original study) show a relative selectivity during the incorporation of nucleotides opposite m 6 A in the RNA template [29]. This observation was used for developing both low-throughput (single-base elongation-and ligation-based qPCR amplification method, termed SELECT protocol) [30] and high-throughput (Locus-specific Extension of Annealed DNA probes targeting m6A and sequencing, LEAD-m6A-seq) approaches [31]. In both of the methods, cDNA extension that is mediated by the Bst DNA polymerase allows for the distinction of unmodified and m 6 A-modified residues at selected loci.…”