Increasing Sensitivity of Next Generation Sequencing-Based Transcriptome Profiling By Selectively Depleting Abundant RNAs in Animal and Plant Samples

High-throughput sequencing (RNA-seq) is now a standard method for large-scale transcript level profiling. Sequencing an entire transcriptome is inefficient since most sequence reads correspond to a few extremely abundant transcripts, so a method to enrich transcripts of interest is typically employed.

We introduce a simple method to selectively degrade unwanted RNAs before sequence library preparation. This method is based on hybridization of probes to the unwanted RNA sequences and subsequent digestion of the targeted RNAs by RNaseH. We target nuclear and organellar RNA components of ribosomes (rRNA) as well as other abundant transcripts (e.g., components of the photosynthetic machinery in plants).

We assessed the performance of the method via extensive directional RNA sequencing. Efficiency was determined through comparison of targeted transcript levels between depleted and untreated samples. High depletion efficiency is achieved (up to 99% rRNA depletion efficiency) even in the context of partially degraded input RNA. Depletion specificity was measured by comparing levels of non-targeted transcripts before and after depletion. Excellent transcript correlation between depleted and non-depleted libraries (r=0.96) demonstrates minimal off target effect. Depleted libraries retain even coverage across the length of transcripts. Both coding and non-coding RNA species are faithfully represented in the treated libraries.

The method is simple, efficient and specifically degrades targeted transcripts. Depleted libraries reflect the diversity of the source RNA and enable a researcher to examine both coding and non-coding transcripts without exhaustive sequencing.