Ribosome Profiling Q&A

Ribosome sequencing can obtain accurate information and precise quantification of all translatable molecules (including mRNA and other potentially translatable RNA molecules such as lncRNA, circRNA, etc.) in a sample, and is a bridge between the transcriptome and proteome.

RNA-Seq can reflect the type and quantity of mRNA in cells, and can also detect sequence variation and variable splicing of mRNA. However, it does not reflect whether mRNA is translated into the corresponding protein and the level of translation. Translatome analysis can study the level, region, and rate of gene translation in cells, etc. Combining transcriptome, small RNA sequencing, and proteome for correlation analysis, we can study the post-transcriptional regulation and translation regulation mechanism more precisely.

Capture translation rates can reveal which genes are being made, in what quantities, and when they are needed. Ribosome profiling allows genome-wide monitoring of ongoing protein synthesis and complements other global approaches (e.g. RNA sequencing). In fact, ribosome sequencing has been used to study the mechanism of action of phenotypic antibiotics in bacteria, the mechanisms of regulation in plants and animals under adversity stress, the molecular mechanisms of developmental aging in individuals, and the mechanisms of tumor development, among others. If you have a project that requires consultation, you can contact our technical experts.

Poly-A tailing method sacrifices the yield to guarantee 5' end accuracy of the library which is critical for good triplet periodicity. The IFR based on this method is approximately 50-60%.
Adaptor ligation method obtains a high yield; however, the 5' end accuracy of the library is low. There are ~30% reads whose first nucleotides cannot be aligned to genome, probably due to the untemplated addition during reverse transcription. Thus, removal of the first mismatch nucleotide from the 5' end is needed to obtain accurate P-site.

The Protein synthesis inhibitor cycloheximide (CHX) is widely used in Ribo-seq experiments. Recent studies have found that CHX may cause transcriptional upregulation of genes involved in ribosome production, resulting in a significant decrease in translation efficiency. In addition to the effect on transcription, the concentration of CHX used also affects the distribution of ribosomal "footprints" on mRNA, with low CHX concentrations causing an artificial bias that can be avoided by increasing CHX concentrations.

Using ribonuclease (RNase) to digest mRNA parts located outside of the ribosome is an important step in ribosome sequencing. Therefore, the selection of RNase and the concentration used are crucial.
Currently, the most commonly used is RNase I. The greatest advantage of RNase I over others is that it has no base preference for cleavage. However, for some species (e.g. human), the efficiency of obtaining 80s ribosomes with RNase I is low, and it is difficult to obtain the desired 80S ribosomes if the amount of RNase I is not well controlled, as shown by the fact that if the concentration of RNase I is too high, the integrity of ribosomes will be severely damaged, making the final library contain a large amount of ribosomal RNA (rRNA). If the concentration of RNase I is too low, although some ribosomal integrity can be maintained, it will make the ribosome-protected mRNA fragments incompletely cleaved. Therefore, a pre-test is required to determine the ribonuclease to be used and the working concentration of the ribonuclease.