For quantification of m6A in poly(A) RNA, nucleoside digestion was performed as previously described(31 (link)). Separation was accomplished by reversed phase chromatography with an Acquity UPLC HSS T3 (Waters) on a Vanquish™ Flex Quaternary UHPLC system (Thermo Fisher Scientific). Mass spectrometry was performed on a Quantiva™ triple quadrupole mass spectrometer interfaced with an H-ESI electrospray source (Thermo Fisher Scientific). Data were analyzed with Tracefinder 4.1 (Thermo Fisher Scientific) and Qual browser of Xcalibur 3.0. The mass transitions (precursor → product) for m6A were 282 → 94, 282 → 123 and 282 → 150.
Changes in m6A were also measured by dot blot from 50ng of poly(A) RNA. Blotting was performed as previously described for 5-hydromethylcytosine(32 (link), 33 (link)), except that Diagenode C15410208 (1:400) was used as primary antibody.
Changes in m6A were also measured by dot blot from 50ng of poly(A) RNA. Blotting was performed as previously described for 5-hydromethylcytosine(32 (link), 33 (link)), except that Diagenode C15410208 (1:400) was used as primary antibody.
