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

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.

Automated summary

High-throughput sequencing was used to analyze the landscape of small noncoding RNAs, revealing a large number of previously undetected sRNAs. The study also reported extensive tissue-specific differences in sRNAs by comparing sRNomes from nine mouse tissues. Additionally, the researchers observed changes in sRNomes during hepatic reprogramming, and found that knockdown of a certain type of sRNA promoted this reprogramming process.