HRA008135
(Controlled Access)
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293T cells were transfected with either an exogenous METTL1 overexpression plasmid or an empty vector control plasmid. At 48 hours post-transfection, cells were subjected to UV crosslinking at 4000*100 µJ/cm2 energy. Cells were then lysed, and anti-FLAG magnetic beads were used for immunoprecipitation of crosslinked RNA-protein complexes for 4 hours at 4°C with rotation. RNA was subsequently extracted from the immunoprecipitated material using TRIzol reagent following the manufacturer's instructions. Recombinant wild-type AlkB and the D135S AlkB mutant proteins were employed to remove dominant methylations from the isolated RNA samples, facilitating efficient reverse transcription of tRNAs. |