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By identifying transcripts undergoing active translation, Ribo-Seq can provide visibility into an important aspect of gene regulation.
What is ribosome profiling?
Ribosome profiling is a method based on deep sequencing of ribosome-protected mRNA fragments. Ribosome profiling, also known as Ribo-Seq (ribosome sequencing) or ART-Seq (active mRNA translation sequencing), provides a “snapshot” of all the ribosomes active in a cell at a specific time point. This information can help researchers determine which proteins are being actively translated in a cell.
Ribosome profiling analysis enables systematic monitoring of cellular translation processes and prediction of protein abundance. Determining what regions of a message are being translated can help define the proteome of complex organisms. In combination with massively parallel high-throughput next-generation sequencing (NGS), ribosome profiling with Ribo-Seq allows detailed and accurate in vivo analysis of protein production.
Advantages of ribosome profiling
Generating sequencing libraries for ribosome profiling
Ribosome profiling protocols often use size-exclusion chromatography. This method streamlines the steps to convert ribosome-protected RNA fragments into libraries compatible with sequencing on Illumina instruments. It is a simple, rapid, and scalable method that does not require special equipment. In contrast, other methods to isolate polysomes and monosomes rely on several hours of ultracentrifugation using either a sucrose gradient or a cushion.
Ribo-Seq protocols and solutions
Various ribosome profiling publications featuring Illumina sequencing instruments are available as a reference. For example, the following Nature Cell Biology publication discusses the use of Ribo-Seq to study early neurodevelopment. The paper includes a Ribo-Seq sample preparation and data analysis protocol and indicates that sequencing was performed on an Illumina NovaSeq 6000 System. In addition, Illumina no longer sells Ribo-Seq library preparation kits, but Ribo-Seq kits optimized for Illumina sequencing are available from other companies such as Immagina Biotech.
Read publication: Ribosome levels and neurodevelopment
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Additional resources
This sequencing method simultaneously quantifies cell surface protein and transcriptomic data within a single cell readout.
User-friendly software tools simplify RNA-Seq data analysis for biologists, regardless of bioinformatics experience.
Ribosome profiling reagent details
Illumina TruSeq Ribo Profile kits (discontinued) were based on the protocol and reagents described in this Nature Protocols paper.
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