The Genomics group has created a robust, optimised pipeline for the creation of sequencing libraries for ribosome profiling projects.
Ribosome Protected Fragments (RPFs) are generated:
1) Digestion of lysate with RNase I.
2)Incubation with rotation for 30 minutes at room temperature.
3)Treatment with SUPERaseIn RNase Inhibitor.
4)Purification using Illustra MicroSpin S-400 HR columns (GE Healthcare).
Libraries are prepared for both whole transcriptome samples and the RPFs.
Aligning sequence reads to the whole genome enables not only the relative abundance of individual mRNAs to be measured, but also the prevalence of exons - annotated and novel - and exon-splicing events, as well as single nucleotide polymorphisms (SNPs) and novel single-base variants.
Sequencing of RPFs measures in vivo translation. By targeting only those mRNA sequences protected by ribosomes, the location of translation start sites can be identified, as well as the distribution of ribosomes on an mRNA and the speed of translation.