High-throughput sequencing of short cDNA tags, or RNA-Seq, has become a staple of genome-wide gene expression studies in plants. RNA-Seq libraries necessarily contain tags that correspond to the mRNA-poly(A) junction, or polyadenylation site, and thus may be mined for data that can help study alternative polyadenylation. This report presents a simple, rapid, and inexpensive method for preparing strand-specific RNA-Seq libraries from varying quantities of total RNA.
The uses of RNA-Seq in plant science are manifest and varied, and the ongoing generation of RNA-Seq datasets should allow considerable data mining opportunities for the study of APA in plants. As the use of RNA-Seq grows, so too will the need for facile and inexpensive methods for the production of RNA-Seq libraries. The protocol described in this report is one such method. It is a variation of methods that capitalize on unique features of reverse transcriptases derived from Moloney Murine Leukemia Viruses (MMuLVs); chief among these features is the propensity of the enzyme to append short, untemplated oligo-C tracts to the 3′ ends of cDNAs, and to switch templates using base pairing between these untemplated C’s and 3′-terminal runs of G on the secondary template. The method detailed in this report has the following features: it generates strand-specific sequence tags without the need for modified nucleotide precursors, it is easily adaptable to different high-throughput sequencing platforms, it allows for extensive, user-defined barcoding and thus for multiplexing of samples at the sequencing step, and it is rapid and involves a minimal number of steps. Pertinent to the last point, modifications such as end-polishing, addition of untemplated tracts using reagents other than reverse transcriptase, and ligations are avoided. Importantly, the method in principle can be used to generate strand-specific RNA-Seq libraries from RNA isolated from bacteria as well as from eukaryotes. Because of these features, and because the cost per library is very low, the method is well suited for teaching as well as research purposes, in undergraduate as well as graduate laboratory settings. (read more…)