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Ribosome Profiling with CNOT1 depletion

ABSTRACT: Ribosome profiling in HEK293 cells with and without the depletion of CNOT1. This allows the determination of translational efficiency and nucleotide resolution of ribosome occupancy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE158141 | GEO | 2021/08/23

REPOSITORIES: GEO

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Ribosome Profiling with CNOT1 depletion

Project description:Ribosome Profiling with CNOT1 depletion

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Project description:Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally abundant, ribosomal RNA sequences in order to save on sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

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Ribosome profiling of HEK293 cells.

Project description:The development of the ribosome profiling (ribo-seq) technique has enabled the measurement of translation at a genome-wide level. There are several variants of the ribosome profiling technique that use different translation inhibitors. The regular ribo-seq utilizes Cycloheximide (CHX), a translation elongation inhibitor to freeze all translating ribosomes. In contrast to CHX, the translation inhibitor lactimidomycin (LTM) and harringtonine (Harr) have a much stronger effect on initiating ribosomes. The use of these inhibitors allows for the global mapping of translating initiating sites (TISs) when they are coupled with ribosome profiling (TI-seq). We have developed a computational tool to detect and/or quantitatively compare translation initiation from TI-seq data, and predict novel ORFs from regular CHX-based ribo-seq data. Two replicates of CHX-based ribo-seq experiments and one Harr based ribo-seq were performed in HEK293 cells for confirming the ORFs predicted using public available data.

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