library_construction_protocol: Mutant AlkB enzyme mix was generated through purification of AlkB wild-type protein (pET30a-AlkB ΔN11) (Addgene), D135S mutant protein (pET30a-AlkB-D135S), and D135T mutant protein (pET28a(+)-AlkB-D135T) (Addgene), which were expressed in BL21(DE3) competent E. coli cells (New England Biolabs Inc). Protein expression was induced by 1 mM isopropyl β-d-1-thiogalactopyranoside with 5 uM iron sulfate for 4 h at 30 °C. Cells were centrifuged and resuspended in Buffer A (10 mM Tris-HCL pH 7.4, 300 mM NaCl, 2 mM CaCl2, 10 mM MgCl2, 2 mM 2-mercaptoethanol, 5% glycerol) containing protease inhibitors. Samples were lysed by sonication. Supernatants were loaded onto a Ni-NTA column (Qiagen) pre-equilibrated with Buffer A and eluted using 250 μM of imidazole. Fractions containing purified AlkB proteins were pooled and dialyzed against storage buffer (30 mM Tris pH 8, 2 mM 2-mercaptoethanol), then supplemented with 30% glycerol and snap frozen. RNA demethylation was performed in a 50 μL reaction volume containing 1 μM purified AlkB (wt, D135S, and D135T) enzymes, 25 mM MES buffer pH 6.0, 2 mM MgCl2, 270 mM KCl, 2 mM L-ascorbic acid, 300 μM α-ketoglutarate, 283 μM (NH4)2Fe(SO4)2, and 1 U/mL SUPERaseIn RNase Inhibitor. Reactions were carried out at 25 °C for 2 h then stopped by the addition of EDTA. RNA was re-purified using RNeasy Mini Kit (Qiagen, 74104) and used as input for sRNA sequencing library preparation. Libraries were prepared using NEXTFlex Small RNA-seq kit v3 (Perkin Elmer) and size-selected using Pippin Prep (Sage). Libraries were quantified with Qubit (Thermo Fisher) and assessed using Bioanalyzer high-sensitivity DNA chips (Agilent). Paired-end sequencing (PE-150) using equimolar concentrations of multiplexed libraries were completed using NovaSeq6000 (S4, Illumina) by the Vanderbilt Technologies for Advanced Genomics Core.