Increasing sensitivity of transcriptome profiling across species by depleting abundant RNAs.

RNA-seq is a widely used technology with a broad range of applications. The technology has been pushed to extremes of very low and degraded samples but still battles with the challenge of having a large dynamic range of transcript expression. Highly expressed transcripts with minimal biological interest can dominate readouts, masking detection of more informative low-abundance transcripts. Here, we present an improved method to enrich for RNAs of interest by eliminating abundant, typically unwanted, RNAs. This method is based on hybridization of probes to the target RNA followed by degradation of the bound RNAs and probes. Optimized probes target rRNAs from human, mouse, rat or bacteria, and globin transcripts from multiple derivate blood samples. Incorporation of new enzymes and streamlining of the method make it more robust with increased specificity. Using strand-specific RNA sequencing we measured sequencing metrics before and after depletion across a wide range of samples, including UHRR, RNA from blood, bacterial monocultures, and communities with 10ng-1µg inputs. Across all samples we achieved high depletion efficiency (up to 99.9 %) with minimal off target effects. We detect a high number of transcripts, with even coverage across the transcript length, while retaining transcript complexity even at the lowest inputs. The method works efficiently with low input and highly degraded total RNA including FFPE samples. We have also developed a customizable approach designed to deplete unwanted RNA from any organism with a user-friendly web tool. Using this tool and method to deplete unwanted RNAs from various species, high depletion efficiency has been achieved for targeted RNAs. We conclude that the reduction of abundant transcripts for RNA-seq studies significantly increases the ability to detect true biological variation that could not be detected in non-depleted samples. The method described here is a reliable and simple solution that greatly improves se.