text: UV-induced RNA-antibody crosslinking was added on top of m6A-seq to produce PA-m6A-seq (photo-crosslinking-assisted m6A-seq) which increases resolution up to ~23nt. First, 4-thiourodine (4SU) is incorporated into the RNA by adding 4SU in growth media, some incorporation sites presumably near m6A location. Immunoprecipitation is then performed on full-length RNA using m6A-specific antibody [36].  UV light at 365 nm is then shined onto RNA to activate the crosslinking to the antibody with 4SU. Crosslinked RNA was isolated via competition elution and fragmented further to ~25-30nt; proteinase K was used to dissociate the covalent bond between crosslinking site and antibody. Peptide fragments that remain after antibody removal from RNA cause the base to be read as a C as opposed to a T during reverse transcription, effectively inducing a point mutation at the 4SU crosslinking site. The short fragments are subjected to library construction and Illumina sequencing, followed by finding the consensus methylation sequence.
  The presence of the T to C mutation helps increase the signal to noise ratio of methylation site detection as well as providing greater resolution to the methylation sequence.
  One shortcoming of this method is that m6A sites that did not incorporate 4SU can't be detected.
  Another caveat is that position of 4SU incorporation can vary relative to any single m6A residue, so it still remains challenging to precisely locate m6A site using the T to C mutation.