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Computational challenges, tools, and resources for analyzing co‐ and post‐transcriptional events in high throughput

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Co‐ and post‐transcriptional regulation of gene expression is complex and multifaceted, spanning the complete RNA lifecycle from genesis to decay. High‐throughput profiling of the constituent events and processes is achieved through a range of technologies that continue to expand and evolve. Fully leveraging the resulting data is nontrivial, and requires the use of computational methods and tools carefully crafted for specific data sources and often intended to probe particular biological processes. Drawing upon databases of information pre‐compiled by other researchers can further elevate analyses. Within this review, we describe the major co‐ and post‐transcriptional events in the RNA lifecycle that are amenable to high‐throughput profiling. We place specific emphasis on the analysis of the resulting data, in particular the computational tools and resources available, as well as looking toward future challenges that remain to be addressed. WIREs RNA 2015, 6:291–310. doi: 10.1002/wrna.1274

Summary of post‐transcriptional regulation processes and corresponding computational methods.
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Read profiles of untreated and harringtonine‐treated RP data. The genic ORF and two uORFs in the Nanog transcript are shown. Start codons are highlighted, and the offset of the 5′ end of reads is indicated.
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(a) Overview of ribosomal profiling (RP) experiments. (b) Detailed steps in the ArtSeq protocol for ribosomal profiling. The protocol starts with cell fragmentation; the resulting cell extract is submitted to nuclease digestion, which will generate ribosome‐protected RNA fragments. Ribosome‐RNA complexes are purified using gel filtration columns (SV400 samples) or sucrose cushion (sucrose samples), followed by RNA extraction and elimination of ribosomal RNAs (rRNA). rRNA‐depleted samples are submitted to electrophoresis, and ribosome‐protected fragments (about 35 nt long) are eluted from gel. These RNAs are used as templates for library preparation and sequencing.
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