High Input rRNA depletion Kit

rRNA Depletion
RNA input range: 5µg - 30µg
RNA quality: RIN > 7
RNA-Seq

High Input riboPOOLs

Ribodepletion kit for total RNA input of more than 5 µg. Removes unwanted ribosomal RNA from total RNA samples, enabling clearer insights for applications like Direct RNA Sequencing or detecting rare or lowly expressed transcripts.

All Available riboPOOLs

Why High Input?

Go to classic riboPOOLs

High Input

Process up to 30µg of total RNA per sample instead of the usual maximum of 3µg.

Shorter Workflow

Consolidate what previously required 10 separate reactions into a single step.

Cost-saving

For depleting large amounts of RNA, this kit will be the cheapest option per-reaction.

When Do You Need High-Input rRNA Depletion?

The High-Input riboPOOL rRNA Depletion Kit (5 µg–30 µg input) is developed for RNA sequencing workflows that require large amounts of depleted RNA.

Typical applications include:

  • Direct RNA sequencing
  • Long-read sequencing
  • Detection of rare or lowly expressed RNAs

After depletion, the RNA is well suited for advanced long-read transcriptomics workflows, including Oxford Nanopore Technologies and Pacific Biosciences (PacBio) platforms. As a result, transcript structures and splice variants can be characterized with greater precision.

Because ribosomal RNA is efficiently removed, sequencing capacity is directed toward biologically relevant transcripts. Consequently, rare and low-abundance RNAs are detected more reliably, and overall data quality improves.

In particular, high RNA input is essential for direct RNA sequencing. Since this approach avoids PCR amplification, more starting material is required to generate sufficient signal and sequencing depth. Moreover, this enables applications such as RNA modification analysis (epitranscriptomics).

How Much RNA Is Considered “High Input” for rRNA Depletion?

The riboPOOL High Input Kit expands our proven rRNA depletion technology to process up to 30 μg of total RNA per reaction. This represents a 10-fold increase compared to standard protocols.

As a result, high-input workflows become more cost-efficient while maintaining the same reliable depletion performance. At the same time, the kit remains fully compatible with short- and long-read sequencing platforms. In addition, it supports the broad species coverage that defines the riboPOOL platform.

Validation Data

Figure 1. Proportion of sequencing reads mapping to rRNA, mRNA, lncRNA, snRNA, and tRNA in A549 cell samples before (non-depleted) and after rRNA depletion using the Human High-Input riboPOOL. Data were generated by direct RNA sequencing using Oxford Nanopore Technologies following in vitro polyadenylation. For depleted samples, analyses were performed either on all retained fragments >17 nt or on a size-selected fraction containing only reads >200 nt. 

Figure 2. Proportion of sequencing reads mapping to different RNA classes in rRNA-depleted samples prepared with or without in vitro polyadenylation. Poly(A) tailing increases the representation of lncRNAs and snRNAs in Oxford Nanopore direct RNA sequencing datasets.

 

Figure 3. Bioanalyzer data after ribodepletion of 7, 14 and 28 μg of depleted total input RNA.

Figure 4. Bioanalyzer profile of human rRNA-depleted RNA, including or excluding the small nucleotide fraction (<200 nucleotides). Column-based purification provides the option to include or exclude the small nucleotide fraction, depending on your needs.

 

Figure 5. rRNA depletion preserves transcript length representation. Mean mRNA lengths before and after depletion showed an almost perfect correlation (R = 0.996, p < 2.2 × 10⁻¹⁶).

Figure 6. GAPDH sequence length in non-depleted and rRNA-depleted A549 cells samples. Data were generated by direct RNA sequencing using Oxford Nanopore Technologies without in vitro polyadenylation

FAQ: Ribodepletion for more than 5 µg total RNA samples

Up to 30 µg of total RNA.

We strongly recommend using a total RNA input between 5 µg and 30 µg for this kit. If you need a kit for a lower input amount, we recommend using our standard riboPOOls.

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

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

For the high input riboPOOL, the recommended purification strategy is to use a column-based product. We recommend the RNA Clean and Concentrator kit (from Zymo Research).

The kit is currently only 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

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