High-Input rRNA Depletion up to 30 µg: Breaking the 5 µg Barrier for RNA-Seq Workflows

Published

Ribosomal RNA (rRNA) depletion is an integral step of many RNA sequencing workflows and is designed to remove highly abundant ribosomal transcripts, which typically constitute over 80–90% of total RNA samples. Because rRNA is often not of interest to researchers, its presence in sequencing libraries can prevent the sensitive and cost-effective detection of lower-expressed RNAs of interest. Ribodepletion is strongly recommended when working with degraded RNA samples or non-polyadenylated transcripts where standard poly-A selection is not an option.

Restricted Capacity of Current rRNA Depletion Methods

 

In most commercial rRNA depletion kits, removal of rRNA typically occurs via antisense probe hybridization followed by either magnetic pull-down (via streptavidin-coated beads) or enzymatic digestion (RNaseH). One limitation of these approaches is the amount of RNA that can be processed, often limited to 5 µg or less. Splitting samples into different aliquots during ribodepletion is technically possible. However, this leads to a significant increase in reagent costs and the risk of variability in the depletion efficiency. This can be very limiting when trying to characterize very lowly-expressed or rare RNAs and for certain downstream applications where a large amount of input RNA is needed. One example is Direct RNA Sequencing (Oxford Nanopore) where the lack of PCR amplification means that every read must come from a native molecule. Depleting rRNA from large amounts of total RNA allows to later achieve sufficient pore occupancy and capture rare isoforms or base modifications.

Introducing the High-Input riboPOOL Kit (Up to 30 µg Total RNA)

 

To overcome this issue, we recently launched a new High-Input riboPOOL kit, which allows to deplete up to 30 µg of input RNA per sample without the need for sample aliquoting or the use of large reagent volumes.

The new kit is compatible with both long and short read sequencing and can be easily integrated in established sequencing workflows.

The core ribodepletion workflow remains the same. First, the riboPOOL biotinylated probes hybridize to their target rRNA. After hybridization, the rRNA is removed from the sample via magnetic streptavidin-coated beads. Post-depletion purification is then carried out using RNA purification columns, instead of SPRI beads or precipitation. This provide multiple advantages: faster workflow, lower cost and smaller reaction volumes. The entire procedure can be completed in less than 90 minutes, allowing researchers to proceed directly to their downstream applications.

The column-based purification also provides the option to split the recovered RNA into large (> 200 nucleotides) and small (< 200 nucleotides) fractions. This can be particularly useful depending on the downstream application, such as when independent analysis of mRNA and small RNA species is required.

Want to know more? You can find more information and the full list of available High-Input riboPOOLs here. Would you like to get in contact with our product specialist or request a quote? You can reach us at info@sitools.de

FAQ

Up to 30 µg of total RNA.

Yes. The kit is compatible with both long and short-read sequencing platforms

The kit is currently available for all our standard RNA-Seq riboPOOLs, which are compatible with RNA samples with RIN value of 7 or more.

The amount depends on type of sample and depletion efficiency. We regularly recover > 400 ng rRNA-depleted RNA when starting from 30 µg

Although technically possible, we recommend using the standard version of riboPOOLs. These are better suited and a more cost-effective solution when depleting smaller RNA amounts.

Yes. We offer custom design for both standard and high-input riboPOOLs.