2. Academic Validation
  3. CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

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

  • Cell Discov. 2021 Apr 19;7(1):25. doi: 10.1038/s41421-021-00265-2.
Heming Wang  # 1 2 3,  Rong Huang  # 1 2 3,  Ling Li  # 1,  Junjin Zhu 1 3,  Zhihong Li 4,  Chao Peng 4,  Xuran Zhuang 1,  Haifan Lin 5,  Shuo Shi 6,  Pengyu Huang 7 8 9
Affiliations

Affiliations

  • 1 School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
  • 2 University of Chinese Academy of Sciences, Beijing, 100049, China.
  • 3 CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China.
  • 4 National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, 201210, China.
  • 5 Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520, USA.
  • 6 Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, 201210, China. shishuo@shanghaitech.edu.cn.
  • 7 School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. huangpengyu@yeah.net.
  • 8 CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China. huangpengyu@yeah.net.
  • 9 Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300192, China. huangpengyu@yeah.net.
  • # Contributed equally.
PMID: 33867522 DOI: 10.1038/s41421-021-00265-2
Abstract

High-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.

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