Commonly used acronym: Ribo-seq
Scope of the method
- Animal health
- Human health
- Translational - Applied Research
- In vitro - Ex vivo
- Human derived cells / tissues / organs
- ribosome profiling footprints
- Gene expression
- cancer research
- Translational Regulation
- human diseases
- Peptide Folding
These are the general guidelines on sample input requirements to successfully start our RIBO-seq procedures:
- Cell lines in suspension: pellet of 10 – 50 M snap frozen cells. The pre-treatment and collection specifications are also mentioned in our pre-processing protocol.
- Adherent cell lines: lysate with minimum concentration of 50 ng/μl (fluorescence measurement: Qubit HS RNA or Ribogreen).
- Hard / Soft tissue types: input amount has to be discussed.
Workflow: *RNase I digestion, sucrose gradient fractionation*, and RNA isolation, as per Supplier’s ribosome profiling protocol *Depletion of rRNA species** (i.e., ribodepletion) *Preparation of ribosome profiling libraries (strand-specific) *Quality control*** (QC) of libraries (e.g., Bioanalyzer analysis or similar, small sequencing run) *Deep sequencing of libraries To measure translation efficiency, normalized ribosomal footprint read counts must be normalized for gene expression. Therefore, total RNA-seq is to be performed. Specifically, CGTC requires: #Total RNA extraction from the same material as that used for ribosome profiling #Ribodepletion #Library preparation for strand-specific total RNA-seq #Quality control (QC) of libraries (e.g., Bioanalyzer analysis, small sequencing run) #Deep sequencing of libraries
iSeq100 Illumina PCR machines Thermomixer
- History of use
- Internally validated
- Published in peer reviewed journal
Pros, cons & Future potential
- - Ribosome profiling is highly beneficial as NGS alternative to or complementary to MS-based protein and peptide identification and will develop into a common practice for next-generation proteomics.
- - With ribosome profiling the translational control is investigated and the gene expression is measured at the translational level.
- - It allows to determine the rate of protein synthesis over a large dynamic range.
- - The price is high.
- - It is an elaborate and sensitive protocol that requires qualified personnel to perform.
- - Limiting factor: High concentration/quantity of material is needed to produce good/ reliable results.
The method can be modified to study Microbiome communities (MetaRibo-Seq) which is a very promising area of research related to human health.
- - Identification of translated sequences within the complex transcriptome,
- - Mapping sites of translation initiation (TIS),
- - Measurement of differential gene expression at the level of mRNA translation,
- - Identification of novel protein coding genes and ribosome pausing.
References, associated documents and other information
- Verbruggen, S., Ndah, E., Van Criekinge, W., Gessulat, S., Kuster, B., Wilhelm, M., ... & Menschaert, G. (2019). PROTEOFORMER 2.0: further developments in the ribosome profiling-assisted proteogenomic hunt for new proteoforms. Molecular & Cellular Proteomics, 18(8), S126-S140.
- Van Damme, P., Gawron, D., Van Criekinge, W., & Menschaert, G. (2014). N-terminal proteomics and ribosome profiling provide a comprehensive view of the alternative translation initiation landscape in mice and men. Molecular & Cellular Proteomics, 13(5), 1245-1261.
Contact personZarel Martinez