5′ Specific RNA-Seq – analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end

Conventional high-throughput RNA sequencing (RNA-seq) uses massively parallel sequencing technology to analyze cDNA derived from total cellular RNA and is usually intended to survey the entire transcriptome. Often, however, it is preferable to derive sequence information from only a particular fraction of the transcriptome. In such cases, methods to generate cDNA libraries derived only from the RNA fraction of interest are required.
Here, researchers from Rutgers University describe a protocol that facilitates the analysis of only the portion of the transcriptome comprising the 5′ ends of RNAs. In particular, they describe a protocol for specifically generating cDNA libraries from RNA 5′ ends. In addition, selective enzymatic treatments of RNA at the beginning of the protocol permits construction of cDNA libraries derived only from transcripts that carry a particular modification at the 5′ end of the transcript. Their lab has developed these protocols for use with bacterial RNAs, which lack a 5′ 7-methylguanosine mRNA cap. However, the methods described here are easily adaptable to the analysis of RNA derived from any organism.

Enzymatic treatments to prepare RNA for cDNA library construction

Vvedenskaya I1, Goldman SR, Nickels BE. (2015) Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends. Methods Mol Biol 1276:211-28. [abstract]

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