Discovering the RNA Transcription Landscape using Directional Approaches

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful technique that allows for sensitive digital quantification of transcript levels, enables the detection of non-canonical transcription start sites as well as termination sites, alternative splice isoforms and transcript mutation and edition. Standard “next-generation” RNA-sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. This work describes a novel streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information and evenly coverage transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differentially tagging of the first strand cDNA ends.  Additional to mRNA sequencing data, whole transcriptome analysis (Total RNAseq) from ribosomal-depleted samples will be presented. Different methods for ribosomal RNA removal, including rRNA reduction by enzymatic degradation will be shown. Total RNAseq provides a much broader picture of expression dynamics including discovery of antisense transcripts. We applied this method to understand the E.coli transcriptome. We found that the method is highly efficient at generating stand specific information.  For the 50 most abundant transcripts the sense strand represented over 99.5% of the reads.  We also discovered novel and existing anti-sense E. coli RNA transcripts using this protocol. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.