200 ng small RNA fraction (<200 nt; mainly composed of mature tRNA) was purified from total RNA using MEGAclear Kit (Thermo). Purified small RNA was deacylated by incubating in 0.1 M Tris-HCl, pH 9 at 37°C for 45 min. Half of the deacylated small RNA fraction was subjected to demethylation treatment by using AlkB in vivo reaction. RNA samples were dephosphorylated with PNK (NEB) and ligated to 3' RNA linker using T4 RNA ligase2, truncated KQ (NEB). Reverse transcription was carried using different reverse transcriptase for different samples. 5' adaptor was ligated to cDNA and then amplified by PCR. PCR products were purified by 8% TBE gel.