![]() ![]() Throughout this work I describe the development of open source computational tools for the analysis and visualisation of CLIP data. Integration of many datasets must be approached carefully, with a view to extract as much biological information as possible. Alongside these experimental developments have come new computational challenges. In particular this thesis focuses on the most abundant internal modification of mRNA, N6-methyladenosine (m6A), and how RNA binding proteins (RBPs) interact with RNA modifications to impact RNA life cycle. In the past decade, leaps forward in biochemistry and high throughput sequencing methods have enabled mapping of RNA modifications across all RNA species. Historically, only abundant modifications on abundant RNAs such as tRNA and rRNA could be studied, due to methodological limitations. The covalent modification of RNA nucleotides is a powerful layer of post-transcriptional control of gene expression across the tree of life. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). ![]()
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