GSM6585998: Old-CMC-2; Mus musculus; OTHER
Small RNA (<200nt) was extracted and purified using miRNeasy Mini Kit (Qiagen) and RNeasy MinElute Cleanup Kit (Qiagen). Purified small RNA fragments were demethylated by wild-type and  mutant AlkBs, purified by phenol/chloroform extraction and ethanol precipitation, labeled by CMC. 50ng small RNA was denatured at 65 °C for 5 min and demethylated by wild-type and D135S mutant AlkBs. The purified small RNA was denatured at 80 °C for 5 min, added to BEU buffer with or without CMC, incubated at 37 °C for 20 min, then purified by ethanol precipitation.The  purified RNA was dissolved in sodium carbonate buffer and shaked at 37 °C for 6 h. The small RNA was dephosphorylated with CIP (NEB). The 3' adaptor ligation was added with T4 RNA ligase2, truncated KQ (NEB), followed by 5' Deadenylase (NEB) and RecJf (NEB) digestion. The RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen), then digested by RNase H. The 5' adaptor ligation was added with T4 RNA ligase 1, high concentration (NEB). The ligated cDNA was amplified by Q5 High-Fidelity 2× Master Mix (NEB).