text: m6A-LAIC-seq (m6A-level and isoform-characterization sequencing) is a high-throughput approach to quantify methylation status on a whole-transcriptome scale. Full-length RNA samples are used in this method. RNAs are first subjected to immunoprecipitation with an anti-m6A antibody. Excess antibody is added to the mixture to ensure all m6A-containing RNAs are pulled down. The mixture is separated into eluate (m6A+ RNAs) and supernatant (m6A- RNAs) pools. External RNA Controls Consortium (ERCC) spike ins are added to the eluate and supernatant, as well as an independent control arm consisting of just ERCC spike in. After antibody cleavage in the eluate pool, each of the three mixtures are sequenced on a next generation sequencing platform. The m6A levels per site or gene could be quantified by the ERCC-normalized RNA abundances in different pools. Since full-length RNA is used, it is possible to directly compare alternatively spliced isoforms between the m6A+ and m6A- fractions as well as comparing isoform abundance within the m6A+ portion.
  Despite the advances in m6A-sequencing, several challenges still remain: (1) A method has yet to be developed that characterizes the stoichiometry between different sites in the same transcript; (2) Analysis results are heavily dependent on the bioinformatics algorithm used to call the peaks; (3) Current methods all use m6A-specific antibodies to tag m6A sites, but it has been reported that the antibodies contain intrinsic bias for RNA sequences.