N6-methyladenosine (m6A) sequencing of messenger RNAs in acute myeloid leukemia (AML) cells with and without knockdown of METTL14 [m6A-seq]

To dissect the mechanism underlying the oncogenic function of METTL14 in AML, we performed m6A RNA immunoprecipitation and deep sequencing for mRNA isolated from MM6 and NB4 cells with and without knockdown of METTL14 Overall design: We lentivirally transduced pLKO.1-based lentiviral shRNAs targeting METTL14 (i.e., shM14-#1 and shM14-#2) or scramble shRNA (i.e., shNS) into human MM6 and NB4 AML cells. After selected with puromycin (0.5 µg/mL) for two passages, cells were collected for genome-wide m6A-sequencing (m6A-Seq) assays. The m6A-seq procedure was performed according to Dominissini's protocol (Dominissini D., et al. Nature 2012; 485: 201-206) with some modifications. Polyadenylated RNA was extracted using the Dynabeads mRNA DIRECT kit (ThermoFisher). RNA fragmentation was performed by sonication at 10 ng/µl in 100 µl RNase-free water using Bioruptor Pico (Diagenode) with 30s on/30s off for 30 cycles. M6A antibody (Synaptic Systems, 202003) was applied for m6A pull down. Library was constructed by TruSeq Stranded mRNA Library Prep Kit (Illumina) and sequenced on Illumina HiSeq 2000.