Sequencing of mRNA in AML cells upon ALKBH5 knockdown and actinomycin D treatment for different time

To evaluate the effects of ALKBH5 KD on the mRNA stability, we conducted RNA-seq of mRNA samples enriched from AML cells with ALKBH5 knockdown and actinomycin D treatment for different time Overall design: AML cells (herein is NOMO1) were transduced with lentivirus expressing shRNAs and selected with puromycin for 4 days. The infected cells were then treated with actinomycin D for different time. Polyadenylated RNAs (poly(A) RNAs) were extracted with FastTrack MAG Maxi mRNA isolation kit (Life technology) and randomly fragmented with RNA fragmentation reagents (Ambion). 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 RNA-seq was conducted on the Illumina HiSeq 2500.