Sequencing of messenger RNAs with N6-methyladenosine modifications in multiple myeloma (MM) with and without forced expression of FTO

We transduced lentivirus harboring vectors expressingGFP-FTO or GFP-control fusion proteins into human multiple myeloma cell line RPMI8226 and then seclected individual stable clones under selection of puromuycin (0.2ug/ml). Two stable lines including FTO-overexpression lines (FTO_OE_8) and control lines (Crtl_FTO_OE_8) were selected for genomewide m6A-sequencing (m6A-seq) assays. The m6A-seq procedure was performed according to a previously reported protocol (Dominissini D., et al. Nat Protocols. 2013; 8: 176-189.). Briefly, mRNA was extracted and purified using VAHTS mRNA Capture Beads (VAHTS, Cat. No. N401-01). ZnCl2 was used to randomly fragment RNA. m6A antibody (202203, Synaptic Systems) was applied for m6A pull down. And A KC-DigitalTM Stranded mRNA Sample Prep Kit (Wuhan Seqhealth Co., Ltd., Cat. No.DR08502) was used for library construction. Then samples were sequenced in an Illumina NovaSeq6000 sequencer.