Ribosome profiling (Ribo-Seq) provides a genome-wide snapshot of protein synthesis by identifying the locations of translating ribosomes at single-nucleotide resolution via deep sequencing of ribosome protected mRNA fragments. First described in 2009, this technique has generated novel insights and a greater understanding of translation processes. However, the time-consuming, labour-intensive protocol, and lack of standardisation has impeded its uptake by the broader research community. Our aim is to modernise the Ribo-Seq approach through incorporation of cutting-edge technology, to develop a protocol at a fraction of the current cost, time, and labour. First, we investigated a number of cell lysis parameters to identify biases and to streamline the harvesting process. We find that the standard cell lysis procedure for Ribo-seq is depleted for a subset of mRNAs encoding cytoskeletal proteins. This issue can be easily resolved by omitting one step of the protocol. The second issue we tackled was the removal of contaminating rRNA. The majority of reads in Ribo-Seq libraries align to rRNA (over 80%), resulting in a reduction of useful mapping reads. We implemented a CRISPR-Cas9 approach to remove rRNA derived sequences from the final libraries. Additionally, we tested a novel approach termed Ribo-FilterOut, which involves EDTA treatment and ultrafiltration of samples to separate the small and large ribosomal subunits and release the mRNA footprints from the complex and reduce rRNA contamination. However, we failed to see an improvement in mapped coding reads or reduction in rRNA contamination. Lastly, a number of optimisations were employed to implement a streamlined Ribo-Seq approach, including adaptation of an existing low-input, one-pot protocol, with removal of critical laborious gel RNA-isolation steps, which helps facilitate high-throughput Ribo-Seq using microfluidic robotics. These modifications reduce the risk of sample loss throughout the procedure and allows for library generation in 2-3 days instead of the standard 5-7 days. As little as 1µg of total RNA input is required, allowing for expansion of ribosome profiling to a wider range of cells and tissues. Additionally, many cDNA library generation protocols exhibit consistent ligation bias at the 5’ end of reads, including our standard approach using SMARTer technology. To circumvent this, we implemented a protocol that uses ordered two-template relay that effectively resolves this heavy CG bias at the 5’ end.

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