Sequencing of messenger RNAs with N6-methyladenosine modifications in acute myeloid leukemia (AML) with and without forced expression of FTO. Homo sapiens

To identify potential mRNA targets of FTO whose m6A levels are affected by FTO in acute myeloid leukemia (AML) cells, we conducted m6A-seq for messenger RNAs isolated from AML cells with and without forced expression of FTO. Overall design: We retrovirally transduced MSCV-PIG-FTO (i.e., human FTO) or MSCV-PIG (i.e., CTRL/Control) into human MONOMAC-6/t(9;11) AML cells and then selected individual stable clones under selection of puromuycin (0.5ug/ml). Four stable lines including two each FTO-overexpressing lines (i.e., FTO+ 1 and FTO+ 2; or FTO_1 and FTO_2) and control lines (i.e., WT 1 and WT 2; or Ctrl_1 and Ctrl_2) were selected for genome-wide m6A-sequencing (m6A-Seq) assays. The m6A-seq procedure was performed as detailed in Dominissini’s method (Dominissini D., et al. Nat Protocols. 2013; 8: 176-189.). Polyadenylated RNA was extracted using FastTrack MAG Maxi mRNA isolation kit (Life technology). RNA fragmentation Reagents (Ambion) was used to randomly fragment RNA. M6A antibody (Synaptic Systems) was applied for m6A pull down. 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.

为鉴定急性髓系白血病（acute myeloid leukemia, AML）细胞中受FTO调控m6A修饰水平的潜在mRNA靶点，我们对过表达或未过表达FTO的AML细胞中分离得到的信使RNA开展了m6A测序（m6A-seq）。总体实验设计：我们将携带人源FTO的MSCV-PIG-FTO载体（即过表达人FTO组）或空载体MSCV-PIG（即对照组/CTRL组）通过逆转录病毒转导至人MONOMAC-6/t(9;11)急性髓系白血病细胞，随后以0.5μg/ml嘌呤霉素筛选获得稳定单克隆。最终选取4株稳定细胞系进行全基因组m6A测序（m6A-Seq）分析，其中包括2株FTO过表达株系（FTO+1、FTO+2，亦可记为FTO_1、FTO_2）以及2株对照株系（WT1、WT2，亦可记为Ctrl_1、Ctrl_2）。本研究的m6A测序操作参照Dominissini等建立的方法（Dominissini D., et al. Nat Protocols. 2013; 8: 176-189.）进行：首先使用FastTrack MAG Maxi mRNA分离试剂盒（Life technology）提取聚腺苷酸化RNA；随后采用RNA片段化试剂（Ambion）对RNA进行随机片段化；使用Synaptic Systems公司的m6A抗体完成m6A富集下拉；最终通过TruSeq Stranded mRNA样本制备试剂盒（Illumina）构建测序文库。文库质量通过BioAnalyzer高灵敏度DNA芯片进行定量，随后在Illumina HiSeq 2500平台上开展深度测序。