CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini


Wang Heming,Huang Rong,Li Ling,Zhu Junjin,Li Zhihong,Peng ChaoORCID,Zhuang Xuran,Lin Haifan,Shi Shuo,Huang PengyuORCID


AbstractHigh-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5′-monophosphate and 3′-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5′ snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.


Ministry of Science and Technology of the People’s Republic of China

National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund


Springer Science and Business Media LLC


Cell Biology,Genetics,Molecular Biology,Biochemistry

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