Strand-Specific Transcriptome Sequencing for Challenging Samples

Next Generation Sequencing (NGS) has empowered a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and dynamic range. A powerful application within this field is stranded RNA-Seq, which is necessary to distinguish closely-related genes and non-coding RNAs ( e.g. lincRNA) or to define genes in poorly annotated, coding-rich genomes, such as many bacteria. Commonly used methods to generate strand-specific RNA-Seq libraries are plagued by protocols requiring several rounds of enzymatic treatments and cleanup steps, making them time intensive, insensitive, and challenging to process several samples simultaneously. Here we present a novel, single tube method, based on Clontech's patented SMART technology, which is able to generate strand-specific RNA-Seq libraries from minute sample quantities in under four hours. This approach eliminates the multitude of labor-intensive enzymatic steps required by other stranded RNA-Seq methods while maintaining the sensitivity and reproducibility characteristic of SMART. We have successfully tested our technology with input levels from 100 pg to 100 ng poly(A)-selected RNA, as well as ribosomally depleted FFPE RNA, with outstanding reproducibility within and across input levels. Spike in of ERCC controls showed linear detection over six orders of magnitude and strand specificity of over 99%. The increased sensitivity achieved from SMART requires a more sensitive rRNA removal method; most methods typically require microgram amounts of total RNA. With this in mind, we have developed a method of rRNA depletion which effectively removes 28S, 18S, 5.8S, 5S, and 12S transcripts from mammalian samples down to 10 ng. The remaining RNA can easily be used in downstream sequencing applications with fewer than 5% of reads mapping back to rRNA. With these tools, researchers can more confidently apply NGS to challenging samples.