High-throughput sequencing for 1-methyladenosine (m¹A) mapping in RNA

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• 作者：Lyudmil Tserovski^a ; ¹ ; Virginie Marchand^b ; ^c ; ¹ ; Ralf Hauenschild^a ; Florence Blanloeil-Oillo^b ; ^c ; Mark Helm^a ; Yuri Motorin^b ; ^c ; ^{Yuri.Motorin@univ-lorraine.fr" class="auth_mail" title="E-mail the corresponding author}
• 关键词：DMS ; dimethylsulfate ; 2D(3D) ; bi (tri) dimensional ; TLC ; thin layer chromatography ; HPLC ; high-pressure liquid chromatography ; MS (MS) ; mass spectrometry (tandem mass spectrometry) ; RT ; reverse transcription ; NGS ; next generation sequencing ; CMCT ; N-cyclohexyl-N&prime ; -(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate ; RF ; Random Forest ; PAGE ; PolyAcrylamide Gel Electrophoresis
• 刊名：Methods
• 出版年：2016
• 出版时间：1 September 2016
• 年：2016
• 卷：107
• 期：Complete
• 页码：110-121
• 全文大小：2488 K

文摘

Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m¹A) residues using high-throughput next generation sequencing (NGS). Since m¹A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types of cDNA products essential for further bioinformatic analysis. We demonstrate that m¹A residues produce characteristic arrest and mismatch rates and combination of both can be used for their detection as well as for discrimination of m¹A from other modified A residues present in RNAs.