Probing N 6-methyladenosine (m6A) RNA Modification in Total RNA with SCARLET

Posttranscriptional N (6)-methyladenosine (m(6)A) RNA modification is indispensable for cell development and viability; however, functional investigation of m(6)A biological function has been hindered by the lack of methods for its precise identification and quantitation. Here, we describe a method that accurately identifies m(6)A position and modification fraction in human messenger RNA (mRNA) and long noncoding RNA (lncRNA) at single-nucleotide resolution, termed as "site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET)" (Fig. 1). This method combines two previously established techniques, site-specific cleavage and splint ligation, to probe the m(6)A RNA modification status at any mRNA/lncRNA site in the total RNA pool.