mRNA sequencing in acute myeloid leukemia (AML) cells with and without knockdown of FTO

To identify the expression of mRNAs after knockdown of FTO, we performed RNA-Seq in MA9.3ITD cells with or without knockdown of FTO. We lentivirally transduced pLKO.1-shFTO (i.e., shFTO) or pLKO.1 empty vertor (i.e., shNS) into human MA9.3ITD (human CD34+ hematopoietic stem/progenetor cells stably infected by MLL-AF9 and FLT3-ITD) AML cells and then selected positively infected cells under selection of puromuycin (0.5ug/ml). The knockdown efficiency was confirmed by qPCR and western. Two stable lines including one FTO-knockdown cell line (i.e., shFTO) and one control line (i.e., shNS) were selected for RNA-Seq. Polyadenylated RNA was extracted using FastTrack MAG Maxi mRNA isolation kit (Life technology). RNA fragmentation Reagents (Ambion) was used to randomly fragment RNA. And final library preparation was constructed by TruSeq Stranded mRNA Sample Prep Kit (Illumina). Final library was quantified by BioAnalyzer High Sensitivity DNA chip then deeply sequenced on the Illumina HiSeq 2500.