library_construction_protocol: Cellular total RNA in TRIzol LS (ThermoFisher #10296028) was extracted by Direct-zol RNA Miniprep kit (Zymo #R2070). mRNA was purified by Dynabeads Oligo (dT)25 (Ambion #61002). Library strategy: GLORI; To study mRNA m6A stoichiometries, we used a modified protocol of GLORI 2.0{Sun, 2025 #15}. mRNA was first purified from 1 µg of total RNA by one round of Poly(A) selection by Dynabeads Oligo (dT)25 (Ambion #61002). mRNA was fragmented into 150-200nt fragments by incubation at 94°C for 3 min with 2µl 10X RNA Fragmentation buffer (NEB E6150S); the reaction was terminated with 2µl 10X stop buffer and cleaned-up by ethanol precipitation. Prior to conversion, samples were denatured at 65°C for 5 min and then stored on ice to prevent secondary structures from impeding conversion. Following the conditions from the GLORI2.0 protocol68, samples were converted in deamination buffer (13μl glyoxal, 10μl DMSO, 10μl 5 M NaNO2, 5μl H3BO3, 4μl 500 mM MES (pH 6.0)) for 10 min at 50°C. Next, samples were ethanol precipitated, and the glyoxal adduct was removed with deprotection buffer (500 mM TEAA pH=8.6, 47.5% deionized formamide, 2mM EDTA) for 10 min at 95°C. RNA was collected again by ethanol precipitation, and end repair was carried out with 1 µl T4 PNK (NEB, M0201L), 2 µl 10X T4 PNK Reaction Buffer, and 1 µl SUPERase·In RNase inhibitor (ThermoFisher #AM2696) for 30 min at 37 °C. The reaction was cleaned up using an RNA Clean & Concentrator-5 column (Zymo Research, R1016), and the RNA was eluted in 10µl water. 5' ligation was carried out with 20 pmol of 5' adapter (see Table S4), 2 µl T4 RNA ligase 1 (ssRNA Ligase, 30U/µl) (NEB, M0437M), 4 µl 10X T4 RNA Ligase Buffer, 4 µl 10mM ATP, 0.5 µl SUPERase·In RNase inhibitor, and 16 µl 50% PEG8000 for 2 hours at 25 °C. 5' ligation product was purified with ethanol-AMPure XP (RNA:beads:ethanol=1:2:3), washed twice in 80% ethanol, and eluted in 10 µl water. 3' adapter incorporation and cDNA synthesis were carried out via a template switching reaction with an R2R RNA/R2R DNA heteroduplex adapter based on TGIRT-seq (see Table S4). The template switching reaction was first pre-incubated at room temperature for 30 min with 2 µl 10X annealed R2 RNA/R2R DNA heteroduplex mix (100 nM final conc.), 1 µl Induro RT (200U/µl) (NEB, M0681L), 4 µl 5X Induro RT Buffer, and 1 µl SUPERase·In RNase inhibitor; next, 1 µl 10mM dNTP was added before incubating for 5 min at 25 °C, 15 min at 55 °C, 1 min at 95 °C, and holding at 4 °C. 5 µl of the cDNA product was carried forward for indexing PCR, mixed with 15 µl water, 25 µl Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531L), 2.5 µl i5 indexing primer, and 2.5 µl i7 indexing primer (NEB, E7780S). Cycling conditions were 98 °C for 30 sec, then 15 cycles of 98 °C 15 seconds, 65 °C 30 seconds, and 72 °C 15 seconds, with a final 5 min 72 °C extension. Two rounds of 0.9X AMPureXP bead purifications were performed to remove primers. 2-20 ng indexed library was obtained for each library. The libraries were pooled, purified one more time by 1.0X SPRIselect beads (Beckman Coulter, B23318), and sequenced by NovaSeqX.