Preparation of RNA: RNAs were prepared by in vitro transcription in 1× transcription buffer [40 mM Tris base, 5 mM dithiothreitol (DTT), 1 mM spermidine and 0.01% Triton-X (pH = 8.5)] with addition of 3–6 mM ribonucleoside triphosphates (NTPs), 10–20 mM magnesium chloride (MgCl2), 30–40 ng/μL DNA template, 0.2 unit/mL yeast inorganic pyrophosphatase (New England Biolabs)[61], ∼15 μM T7 RNA polymerase and 10–20% (v/v) dimethyl sulfoxide (DMSO). Reaction mixtures were incubated at 37 °C for 3–4 h, with shaking at 70 rpm, and then quenched using a solution of 7 M urea and 500 mM ethylenediaminetetraacetic acid (EDTA), pH = 8.5. Reactions were boiled for 3 min and then snap cooled in ice water for 3 min. The transcription mixture was loaded onto preparative-scale 10% denaturing polyacrylamide gels for purification. Target RNAs were visualized by UV shadowing and gel slices with RNA were excised. Gel slices were placed into an elutrap electroelution device (The Gel Company) in 1X TBE buffer. RNA was eluted from the gel at constant voltage (120 V) for ~24 h. The eluted RNA was spin concentrated, washed with 2 M high-purity sodium chloride, and exchanged into water using Amicon-15 Centrifugal Filter Units (Millipore, Sigma). RNA purity was confirmed on 10% analytical denaturing gels. RNA concentration was quantified via UV-Vis absorbance. Dimethyl sulfate (DMS) modification of in vitro-transcribed RNA: 3 μg of pre-miR-31-tail RNA was denatured at 95 ℃ for 1 min and incubated on ice for another 3 min. Refolding buffer (300 mM sodium cacodylate and 6 mM MgCl2) was added to reach total volume of 97.5 uL (for the 0% control), 97.5 μL (for 2.5% modified sample) or 95 μL (for 5% modified sample). The RNA was incubated in refolding buffer at 37 ℃ for 40 min. The RNA was treated with either 2.5 µL DMSO (0% DMS), 2.5 μL DMS (2.5% DMS) or 5 μL DMS (5% DMS) followed by incubation at 37 ℃ while shaking at 250 rpm for 10 min. 60 μL β-mercaptoethanol was added to each reaction to neutralize the residual DMS. The modified RNA was purified using RNA Clean and Concentrator-5 kit (Zymo) according to manufacturer's instructions. RT–PCR with DMS-modified RNA:  0.2 μM DMS-modified RNA, 2 μl 5× first strand buffer (ThermoFisher Scientific), 1 μl 10 μM reverse primer, 1 μl dNTP, 0.5 μl 0.1 M DTT, 0.5 μl RNaseOUT and 0.5 μl TGIRT-III (Ingex) were mixed. The mixture was incubated at 57 ℃ for 30 min. After the 30 min incubation, the temperature was increased to 85 ℃ for 5 min. 1 μL RNase H (New England Biolabs) was added to the mixture to digest the RNA. PCR amplification was achieved using Phusion (NEB) for 27 cycles according to the manufacturer's instruction. The PCR product was purified by GeneJET PCR purification kit (Thermoscientific). DMS-MaPseq