repository of small ORFs identified by ribosome profiling

This section provides a summary on the acquisition and processing of the data. For more detailed information, feel free to consult the available literature. Use the navigation bar on the left to navigate trough the different steps.

1. Sequencing

Advances in next generation sequencing allowed techniques, such as ribosome profiling, to emerge. Ribosome profiling (RIBO-seq) captures and subsequently sequences the +/-30bp RNA- fragments captured within ribosomes (the protein translation machinery). This technique differs from a regular RNA-seq setup, as a 'snap-shot' is provided of what is currently being translated in a cell, rather than what is expressed in a cell. In this context, it provides the opportunity to detect small open reading frames (sORF) that are being translated and possibly could encode functional peptides or small proteins. The figure below provides an overview of the RIBO-seq procedure

RIBO-SEQ overview: First, cell lysates are prepared in conditions accurately reflecting in vivo translation. Secondly, addition of nucleases will digest RNA (nuclease footprinting), however the +/-30 nt mRNA fragments encapsulated by ribosomes are protected from digestion (ribosome footprints). Next, ribosome-footprints are separated from cell lysates followed by purification of ribosome protected RNA. Ligation of single-stranded adaptors enables reverse transcription. Subsequently, first strand reverse transcription products are circularized and transcript products hybridized to rRNA probes are depleted. Finally, PCR amplifies the remaining sequences that are subsequently sequenced. An in depth description of the protocol is provided by Ignolia et al 2012.