Sequencing of mRNA with N6-methyladenosine (m6A) modification in AML cells upon ALKBH5 knockdown or overexpression

To identify the potential mRNA targets of ALKBH5, we conducted m6A-seq with mRNA samples enriched from AML cells with ALKBH5 knockdown or overexpression AML cells (herein is NOMO1) were transduced with lentivirus expressing shRNAs or ALKBH5 CDS, then selected with puromycin for 2 days. Polyadenylated RNAs (poly(A) RNAs) were extracted with FastTrack MAG Maxi mRNA isolation kit (Life technology) and randomly fragmented with RNA fragmentation reagents (Ambion). m6A antibody (Synaptic Systems) was used to immunoprecipitate m6A RNA. Final library preparation was constructed with TruSeq Stranded mRNA Sample Prep Kit (Illumina) and the library was quantified by BioAnalyzer High Sensitivity DNA chip. The m6A-seq was conducted on the Illumina HiSeq 2500.