Primers with fam labeling

DNA primers, primers with FAM labeling and U15 RNA for in vitro assays and cloning were ordered from Integrated DNA Technologies, Inc (IDT) with standard desalting. Ligation adaptors used in m¹A-IP-seq and m¹A-IP-seq were ordered from IDT with HPLC purification. Other RNA oligonucleotides used in this study were synthesized in-house using an Expedite DNA synthesizer followed by normal deprotection for regular oligonucleotides and vendor-suggested deprotection for RNA oligonucleotides containing m¹A modifications to avoid Dimroth rearrangement. After deprotection, the RNA oligonucleotides were purified by HPLC with a C18 column and eluted with 0–20% acetonitrile in 0.1 M triethylammonium acetate. The desired peak was collected and dried by lyophilization. Synthesized RNA was dissolved in 10 mM Tris-HCl pH = 7.5, and the quality was examined by 10% 8M urea polyacrylamide gel electrophoresis (PAGE) gel. 33mer A15, m¹A15, and m¹A18 showed decent purity and were used directly. 43-mer RNAs in the spike-in samples showed significant impurity bands so we performed gel purification for all these RNAs with 10% urea PAGE gel and RNA recovery with the ZR small-RNA PAGE Recovery kit (Zymo Research).