m6Aseq

m6A-seq, or m6A sequencing, is a high-throughput method used to map N6-methyladenosine (m6A) residues across transcriptomes. It relies on immunoprecipitation with anti-m6A antibodies to enrich methylated RNA fragments from fragmented total RNA, followed by next-generation sequencing and computational analysis to identify m6A-enriched regions. The approach enabled the first genome-wide maps of m6A in eukaryotic mRNA and has since become a standard tool for studying RNA modification dynamics.

A typical m6A-seq workflow involves isolating RNA, fragmenting it to about 100–200 nucleotides, and performing immunoprecipitation with an anti-m6A antibody. The enriched RNA is purified, converted to cDNA, and sequenced. Sequencing reads are aligned to a reference genome, and peak-calling is performed to identify regions significantly enriched in the immunoprecipitated sample relative to input RNA. Downstream analyses annotate peaks relative to gene features and assess sequence context, often noting enrichment of the DRACH motif (D=A/G/U, R=A/G, H=A/C/U). Conventional m6A-seq provides peak-level resolution on the order of hundreds of nucleotides, with higher-resolution methods available.

Limitations of m6A-seq include dependence on antibody specificity and potential biases in immunoprecipitation efficiency, which can affect sensitivity and accuracy. The technique is semi-quantitative and does not readily measure modification stoichiometry at individual sites without additional methods. Advances such as crosslinking-based methods (e.g., miCLIP, m6A-CLIP) improve resolution to single-nucleotide precision. m6A-seq has been applied to study developmental processes, stress responses, and disease-associated changes in m6A patterns, contributing to the understanding of RNA regulation.