Quantification of m6A in Poly(A) RNA

Total RNA of DC was isolated with Trizol reagent (Invitrogen), and mRNA was enriched by Dynabeads mRNA Purification Kit (Invitrogen). Removal of ribosomal RNA was confirmed by 2200 Tape Station detection (Agilent). In total, 2.5 μl of 10x Reaction Buffer (20 mM of ZnCl₂, 100 mM of NaCl) and 1 μl of Nuclease P1 (1.2 U/μl) (Sigma) were added to 350 ng of purified mRNA and incubated at 37 ℃ for 2 h after adding H₂O to a total volume of 2 μl. Then 2.5 μl of CIAP Buffer and 1 μl of CIAP (Promega) were added and incubated at 37 ℃ for another 2 h. The mix was diluted with H₂O to 100 μl and filtered through a 0.22-µm filter (4 mm in diameter) (Nalgene) and then loaded to a C18 reverse-phase column coupled online to Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified using the nucleoside to base on mass transitions of 268–136 (A), and 282–150 (m⁶A). A standard curve was obtained from pure nucleoside standards running at the same batch of samples. The m⁶A/A ratio in poly(A) RNA was quantified based on the calculated concentrations^{13 (link)}.