DM-tRNA-seq Protocol with Methylation Removal

DM-tRNA-seq was performed following the previously reported protocol [27] (link), [47] (link) with some modifications. Small RNAs (< 200 nt) were first purified using the Quick-RNA Microprep kit (Catalog No. R1050, Zymo Research, Orange, CA). Isolated small RNAs were treated with recombinant wild-type and D135S AlkB proteins to remove the dominant methylations on RNAs. Then, demethylated RNAs were purified with Oligo Clean & Concentrator kit (Catalog No. D4060, Zymo Research). After that, AlkB-treated RNA libraries were constructed with NEBNext Small RNA Library Prep Set (Catalog No. E7330S, New England Biolabs, Ipswich, MA). The cDNA libraries were sequenced on Illumina HiSeq X10 with paired-end 2 × 150 bp read length.