Ribosome Profiling riboPOOLs
Optimized rRNA Depletion for Ribo-Seq
riboPOOL Product Range – Kits for Every Species and Need
A. castellanii Ribo-Seq
A. thaliana Ribo-Seq
Danio rerio Ribo-Seq
D. melanogaster Ribo-Seq
E. siliculosus Ribo-Seq
Escherichia coli Ribo-Seq
Caenorhabditis elegans Ribo-Seq
Human Ribo-Seq
Human rRNA-tRNA Ribo-Seq
Human tRNA Ribo-Seq
H/M/R Ribo-Seq
L. mexicana Ribo-Seq
Mouse-Rat Ribo-Seq
Mouse tRNA Ribo-Seq
P. pacificus Ribo-Seq
S. cerevisiae Ribo-Seq
S. aureus Ribo-Seq
Toxoplasma gondii Ribo-Seq
Trypanosoma brucei Ribo-Seq
What Is Ribosome Profiling (Ribo-Seq)
Ribosome profiling, (or Ribo-Seq), is a Next-Generation Sequencing (NGS) method that involves the isolation and sequencing of ribosome-protected fragments (RPFs). These fragments, approximately 30 nucleotides long, correspond to sections of messenger RNAs (mRNAs) that are found within the ribosome and are protected by it at any given time. By sequencing RPFs, researchers can capture a snapshot of ongoing translation, thereby obtaining a quantitative overview of protein synthesis.
Ribo-Seq applications include:
- Quantifying protein synthesis and translation efficiency
- Identifying novel open reading frames (ORFs) and translation start sites
- Comparing transcription and translation rates to uncover gene regulation mechanisms
Ribosome Profiling Protocol: Step-by-Step Workflow
The Ribo-Seq protocol consists of several critical steps before sequencing. Below is a streamlined overview of each phase, with practical guidance and how our riboPOOLs support the workflow.
Step 1: Cell Harvesting and Lysis
Obtaining a high-quality cell lysate is the first step in ribosome profiling. Cells must be rapidly harvested (e.g., flash-frozen) to preserve ribosome positions on mRNA. Lysis is performed under cold, detergent-containing conditions that stabilize ribosome-mRNA complexes while minimizing RNA degradation. The resulting lysate should be cleared of debris by centrifugation and kept on ice to maintain ribosome integrity, providing a clean starting material for subsequent nuclease digestion and ribosome isolation.
Step 2: RNA Digestion (RNase Treatment)
RNA digestion is a critical step in Ribo-Seq, as it defines the RPFs that will be sequenced. This involves treating the lysate with a ribonuclease—most commonly RNase I—which cleaves non-protected single-stranded RNA, leaving behind only the ~28–30 nucleotide regions physically shielded by the ribosome. Choosing the right RNase is essential: enzymes with biased cleavage or excessive activity can either under-digest (leaving long fragments) or over-digest (damaging RPFs or ribosomes). Optimization of several parameters – including enzyme concentration, incubation time, temperature, and buffer conditions – is thus crucial to ensure complete but controlled digestion.
Step 3: Ribosome Isolation via Sucrose Gradient
To isolate ribosomes and enrich for RPFs, the lysate can be layered onto a sucrose gradient, typically ranging from 10% to 50% sucrose. This gradient allows for the separation of ribosomal complexes based on their size and density through ultracentrifugation. As the sample moves through the gradient during centrifugation, larger complexes such as polysomes or disomes sediment further than smaller particles. Of particular interest in ribosome profiling are the monosomes, which contain a single ribosome bound to an mRNA fragment. These are enriched in RPFs and can be identified by monitoring the gradient with UV absorbance. The monosome (or di-polysome) peak is collected by fractionation of the gradient, typically using a piston gradient fractionator or a needle-puncture (siFractor) system. Following this, the RNA is extracted from the ribosome-containing fractions for further purification and sequencing.
Step 4: Ribosome-Protected Fragment (RPF) Enrichment
Following the isolation of monosome-associated RNA, the RPFs must be purified from other RNA species, such as degraded rRNA, tRNA fragments, and incomplete digestion products. This is typically achieved through denaturing polyacrylamide gel electrophoresis (PAGE), which separates RNA molecules based on size with single-nucleotide resolution under denaturing conditions (usually using 15%–17% urea-PAGE). The denaturing environment ensures that secondary structures in the RNA do not affect migration, enabling accurate size selection. After electrophoresis, the gel is visualized—often using UV shadowing or fluorescent scanning depending on the labeling method—and the precise region corresponding to the RPF size is carefully excised. The excised gel slice is then subjected to RNA elution, typically via passive diffusion or crush-and-soak methods, followed by ethanol precipitation.
A critical step in this process is the precise excision of the ~28–30 nucleotide band corresponding to the RPFs. Accurate excision is essential because contamination with slightly larger or smaller fragments can include unwanted RNA species that do not reflect genuine ribosome-protected regions—potentially skewing downstream sequencing data and reducing resolution. To facilitate accurate identification of the RPF band, specialized RNA (RiboCut Marker) ladders are used. These markers must be carefully chosen to include RNA fragments of known lengths that closely bracket the expected size range of RPFs. A poor choice of ladder can lead to misidentification of the correct band, causing the inclusion of off-target fragments or exclusion of true RPFs.
Step 5: rRNA Depletion with Ribo-Seq riboPOOLs
Despite careful size selection of RPFs, a significant challenge in ribosome profiling remains the co-purification of ribosomal RNA (rRNA) fragments that fall within the same size range. Because of their abundance and similarity in size, these contaminating rRNA fragments can be inadvertently excised and co-sequenced, leading to a substantial proportion of sequencing reads being mapped to rRNA instead of mRNA, thereby reducing the yield of informative data. To address this, ribodepletion strategies are employed post-excision to selectively deplete rRNA contaminants. Incorporating this step is critical for maximizing data quality and sequencing efficiency, ensuring that the majority of reads reflect true ribosome-mRNA. Our Ribo-Seq riboPOOLs are specifically designed to deplete rRNAs from Ribo-Seq samples and significanlty increase RPFs reads mapping rates. Check out our Ribo-Seq riboPOOLs portolio below or request a custom design for your species of interest.
Step 6: Library Generation & Sequencing
After ribodepletion, the RPFs can be converted into sequencing-ready libraries through a series of enzymatic steps. First, a 3' adapter is ligated to the RNA fragments, followed by reverse transcription to generate cDNA. A 5' adapter is then ligated to the cDNA, or alternatively, template switching can be used to add the 5' end. The resulting cDNA is PCR-amplified using indexed primers to allow multiplexing, and the final library is size-selected—often via another round of PAGE or bead purification—to remove adapter dimers and undesired products. The resulting libraries are quantified and assessed for quality before high-throughput sequencing.
rRNA Contamination: Why It Happens
The presence of rRNA constitutes a common characteristc in Ribo-Seq samples, irrespective of the source material, investigated species, or experimental parameters. Although the precise mechanisms behind the emergence of rRNA contaminants remain partially unelucidated, the formation of rRNA contaminants can be ascribed to the activities of ribonucleases used as part of the experiment. Also referred to as RNAses, ribonucleases represent a group of enzymes responsible of catalyzing the degradation of RNAs. In Ribo-Seq experiments, the addition of RNAses is required to cleave and digest all mRNA regions not shielded by ribosomes, while preserving RPFs intact. The ribonuclease digestion represents a critical step, and the selection of the appropriate RNase requires evaluation based upon the characteristics of the source material. During the digestion step however, RNAses also target the rRNAs consituting the ribosomes. This leads to widespread formation of rRNA fragments that are subsequenctly co-isolated with the RPFs. Although RNAses produce rRNA fragments of different length, only contaminants with length comparable to that of RPFs (ca. 30 nt) remain after samples size-selection, and end up in the final libaries.
The effect on rRNA contamination of the size-selection step can be readily seen when plotting reads coverage plots (Figure 3). Here, each rRNA contaminant is represented by a sharp peak with width of ca. 30 nucleotides.
How riboPOOLs Work – Efficient rRNA & tRNA Depletion
Ribo-Seq riboPOOLs are comprised of complex pools of biotinylated probes complementary to their target rRNAs. These probes cover the entire ribosomal RNA sequence, thereby ensuring that any potential contaminant sequence is effectively targeted by a corresponding riboPOOL probe. Abundance of each riboPOOL probe has also been optimized, with probes that target known abundant contaminants being present at higher concentrations.
Pull-down of rRNAs is accomplished through streptavidin-coated beads. Following this step, the purification of rRNA-depleted samples can be achieved using various methods such as ethanol precipitation or the utilization of third-party solutions like the Zymo RNA Clean & Concentrator Kits. It is important to note that the use of SPRI clean-up beads is strongly discouraged due to their potential to result in extensive loss of ribosome-protected fragments (RPFs).
Adding a Ribo-Seq riboPOOLs depletion step into established Ribo-Seq workflows is straightforward. It is advised to carry out rRNAs depletion after size-selection. However, ribodepletion can be performed at any stage of the protocol in which samples consist of purified RNA if required.
Performance & Results – What to Expect with riboPOOLs
Ribo-Seq riboPOOLs efficiently remove rRNAs from ribosome profiling RNA samples. After ribodepletion with the Human Ribo-Seq riboPOOL, RPFs reads mapping rates have been shown to increase by more than 300%.
Depletion of transfer RNA (tRNAs)
Transfer RNAs (tRNAs) can also constitute a significant fraction of Ribo-Seq samples, and are often the most abundant RNAs after ribodepletion. Therefore it is often beneficial to deplete tRNAs alongside rRNAs. The Human tRNA and Mouse tRNA riboPOOLs have been developed to deplete tRNAs from Ribo-Seq samples and further enrich for RPFs in Ribo-Seq samples. The tRNA riboPOOLs can be directly mixed with any Ribo-Seq riboPOOL, allowing for rapid simultaneous depletion of both rRNA and tRNAs.
