Optimized GLORI-Seq of Chromatin-Associated RNAs

N6-methyladenosine (m⁶A) is a prevalent endogenous modification in mammalian mRNAs as well as chromatin-associated RNAs (caRNAs). Recently developed GLORI-seq, which chemically converts unmodified adenosine to inosine, enables absolute quantification of single-base-resolution m⁶A sites on mRNAs. However, since GLORI-seq generates short-transited reads with mainly three kinds of nucleotides, applying it to caRNAs with a large fraction of intronic and intergenic regions poses additional challenges. In our recent study, we simply optimized the library construction of GLORI-seq to enrich long cDNA fragments for subsequent sequencing, resulting in significantly promoted read length and mapping accuracy of GLORI-seq on caRNAs. Here, we describe the detailed protocol of optimized GLORI-seq of caRNAs to facilitate studies on m⁶A in caRNAs.