text: In 2012, the first two methods for m6A sequencing came out that enabled transcriptome-wide profile of m6A in mammalian cells. These two techniques, called m6A-seq and MeRIP-seq (m6A-specific methylated RNA immunoprecipitation), are also the first methods to allow for any type of RNA modification sequencing. These methods were able to detect 10,000 m6A peaks in the mammalian transcriptome; the peaks were found to be enriched in 3’UTR regions, near STOP codons, and within long exons.
  The two methods were optimized to detect methylation peaks in poly(A)+ mRNA, but the protocol could be adapted to profile any type of RNA. Collected RNA sample is fragmented into ~100-nucleotide-long oligonucleotides using a fragmentation buffer, immunoprecipitation with purified anti-m6A antibody, elution and collection of antibody-tagged RNA molecules. The immunoprecipitation procedure in MeRIP-Seq is able to produce >130fold enrichment of m6A sequences. Random primed cDNA library generation was performed, followed by adaptor ligation and Illumina sequencing. Since the RNA strands are randomly chopped up, the m6A site should, in principle, lie somewhere in the center of the regions to which sequence reads align. At extremes, the region would be roughly 200nt wide (100nt up- and downstream of the m6A site).
  When the first nucleotide of a transcript is an adenosine, in addition to the ribose 2’-O-methylation, this base can be further methylated at the N6 position.
  m6A-seq was confirmed to be able to detect m6Am peaks at transcription start sites. Adapter ligation at both ends of RNA fragment results in reads tending to pileup at the 5’ terminus of the transcript. Schwartz et al. (2015) leveraged this knowledge to detect mTSS sites by picking out sites with a high ratio of the size of pileups in the IP samples compared to input sample. As confirmation, >80% of the highly enriched pileup sites contained adenosine.
  The resolution of these methods is 100-200nt, which was the range of the fragment size. 
  These two methods had several drawbacks: (1) required substantial input material, (2) low resolution which made pinpointing the actual site with the m6A mark difficult, and (3) cannot directly assess false positives.
  Especially in MeRIP-Seq, the bioinformatics tools that are currently available are only able to call 1 site per ~100-200nt wide peak, so a substantial portion of clustered m6As (~64nt between each individual site within a cluster) are missed. Each cluster can contain up to 15 m6A residues.
  In 2013, a modified version of m6A-seq based on the previous two methods m6A-seq and MeRIP-seq came out which aimed to increase resolution, and demonstrated this in the yeast transcriptome. They achieved this by decreasing fragment size and employing a ligation-based strand-specific library preparation protocol capturing both ends of the fragmented RNA, ensuring that the methylated position is within the sequenced fragment. By additionally referencing the m6A consensus motif and eliminating false positive m6A peaks using negative control samples, the m6A profiling in yeast was able to be done at single-base resolution.