High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70 % of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs.
Here, scientists at the University of Massachusetts Medical School, describe a procedure for preparing RNA-Seq libraries from 1 to 4 mug total RNA without poly(A) selection. Thier method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. They barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms.
The RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90 % mappable sequences. Typically, more than 85 % of mapped reads correspond to protein-coding genes and only 6 % derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.
Zhang Z, Theurkauf WE, Weng Z, Zamore PD. (2012) Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence [Epub ahead of print]. [abstract]