Identification of ALKBH5 targets in osteosarcoma by methyl RNA immunoprecipitation [MeRIP-seq]

To identify potential target mRNAs that are demethylated by ALKBH5, meRIP-seq with a m6A antibody was conducted in 143B osteosarcoma cells transfected with scrambled or ALKBH5-siRNA. 143B cells were grown in 150 mm tissue culture plates and transfected with with either scrambled-siRNA or ALKBH5-siRNAs (Sigma-Aldrich) for 48 hours.Total RNA / mRNA isolated from the 143B cells was fragmented and subjected to immunoprecipitation (IP) using rabbit anti-m6A antibody. After incubation and washing , samples were eluted using elution buffer containing m6A nucleoside . The resultant final product was used for RNA-seq.