Ribosome Profiling

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.

Most Ribo-Seq protocols consists of four major steps:

  • RNA and RPF preparation
  • rRNA depletion        
  • Library preparation & sequencing
  • Data analysis 

Ribo-Seq applications include:

  • Quantitative assessment of protein synthesis and abundance.
  • Identification of novel open-reading frames (ORFs) and determination of translation initiation sites.
  • Integration with transcriptomics to compare transcription and translation rates, enabling the examination of potential gene regulation mechanisms.

Similar to traditional RNA-Seq, the predominant fraction of Ribo-Seq samples often comprises ribosomal RNA (rRNA), with RPFs (desirable reads) typically representing less than 5% of the sequenced reads. Consequently, the removal of rRNA from samples, prior to sequencing, can:

  • Enhance the cost-efficiency of sequencing projects.
  • Facilitate the quantification of translation of rare transcripts.

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. 

A. castellanii Ribo-Seq
ID: 99

A. thaliana Ribo-Seq
ID: 105

Danio rerio Ribo-Seq
ID: 101

D. melanogaster Ribo-Seq
ID: 76

Human rRNA-tRNA Ribo-Seq
ID: 113

H/M/R Ribo-Seq
ID: 50

L. mexicana Ribo-Seq
ID: 78

Mouse.-Rat Ribo-Seq
ID: 52

E. siliculosus Ribo-Seq
ID: 100

Escherichia coli Ribo-Seq
ID: 104

Caenorhabditis elegans Ribo-Seq
ID: 67

Human Ribo-Seq
ID: 42

P. pacificus Ribo-Seq
ID: 82

S. cerevisiae Ribo-Seq
ID: 49

Toxoplasma gondii Ribo-Seq
ID: 83

Trypanosoma brucei Ribo-Seq
ID: 77

Ribo-Seq riboPOOLs Performance

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%.

Figure 1 a) Proportion of reads mapping to rRNA, tRNA, mRNA and other RNAs in HEK293T samples non-depeleted (left) and depleted with Human Ribo-Seq riboPOOL (right). b) Percentage increase of mRNA reads after depletion with Human Ribo-Seq riboPOOL and competitors products.

How do Ribo-Seq riboPOOLs work

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.

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 direclty mixed with any Ribo-Seq riboPOOL, allowing for rapid simoultaneous depletion of both rRNA and tRNAs. 

Figure 2 Proportion of reads mapping to different RNA types in Ribo-Seq samples after depletion with Human Ribo-Seq riboPOOL only (left), and Human Ribo-Seq riboPOOL in combination with the Human tRNA riboPOOL (right).

The Origin of rRNA Contamination

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. 

Figure 3 Coverage plot of Human 18S rRNA, showing the peaks representing rRNA contaminants. Source material: HepG2 cells