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Ribosome profiling

Most of our current knowledge about gene regulation is based on studies of mRNA levels, despite both the greater functional importance of protein abundance, and evidence that post-transcriptional regulation is pervasive. However, understanding the molecular basis of regulatory variation within and between species may prove very useful. Indeed, the majority of identified human disease-risk alleles lie in non-coding regions of the genome, suggesting that they affect gene regulation. Until recently, the lack of performant high-throughput methods for detecting protein abundance hampered the in-depth study of gene regulation. However, a new method known as ribosome profiling has enabled us to study divergence in the regulation of translation.

Ribosome profiling workflow

The ribosome profiling workflow

Ribosome profiling relies on deep sequencing of ribosome footprints—these short (typically, ∼30 nucleotide) fragments of mRNA that are physically enclosed by the ribosome and shielded from nuclease digestion.

These footprints are converted into a library of DNA fragments and analyzed by one of our Illumina sequencers. The technical quality of the sequencing run is monitored in real time.

Each sequenced footprint reports on the position of one ribosome, revealing what transcript that ribosome was translating and where along the coding sequence it was captured during cell lysis, often with single-nucleotide resolution.

Current deep-sequencing technologies analyze hundreds of millions of individual short reads in one experiment. When applied to libraries of ribosome footprints, this sequencing yields a comprehensive view of the translational landscape that can address many fundamental questions about translations.

Several specific antibiotic treatments during sample preparation result in genome-wide maps of elongating and initiating ribosomes to help delineate the ORF by also capturing the translation initiation events.

We request between 25 – 50 million cells per sample and provided to us as a flash frozen cell pellet. We perform cell lysis ourself

Raw sequencing data can be transferred to you via the server (FTP download) or the Illumina BaseSpace platform.