Fractionation of Non-polyadenylated and Ribosomal-Free RNAs from Mammalian Cells
Most of mRNAs and well-characterized long noncoding RNAs are shaped with 5′ cap and 3′ poly(A) tail. Thereby, conventional transcriptome analysis typically involved the enrichment of poly(A)+ RNAs by oligo(dT) selection. However, accumulated lines of evidence suggest that there are many RNA transcripts processed in alternative ways, which largely failed to be detected by oligo(dT) purification. Here, we describe an enrichment strategy to purify non-polyadenylated (poly(A)−/ribo−) RNAs from total RNAs by removal of poly(A)+ RNA transcripts and ribosomal RNAs. In the combination with high-throughput sequencing methods, this strategy has been successfully applied to identify the rich repertoire of non-polyadenylated RNAs in vivo.
Key wordsRNA fractionation Non-polyadenylated RNAs Long noncoding RNAs Deep sequencing
We are grateful to H.-H. Fang and other lab members for helpful discussion to improve this protocol. This work was supported by grants XDA01010206 and 2012OHTP08 from CAS, and 31271376 and 31271390 from NSFC to LLC and LY.