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[E-MTAB-341] Individual-nucleotide resolution CLIP using high throughput sequencing of human HeLa cells to enable precise mapping of protein-RNA interactions in intact cells

ABSTRACT: UV-crosslinking and immunoprecipitation (CLIP) combined with high-throughput sequencing was previously used to generate transcriptome-wide binding maps of several RNA-binding proteins. However, since identification of binding sites relied on the analysis of overlapping sequence clusters, distances of less than 30 nucleotides were not resolved. An additional disadvantage of CLIP is the requirement of reverse transcription to pass over residual amino acids that remain covalently attached to the RNA at the crosslink site. Primer extension assays have shown that the vast majority of cDNAs prematurely truncate immediately before the 'crosslink nucleotide'. Here, we exploited this apparent limitation to achieve single nucleotide resolution by capturing these truncated cDNAs through the introduction of a second adapter after reverse transcription via self-circularization. In order to quantify cDNA molecules that truncate at the same nucleotide, we added a random barcode to the DNA adapter. This allowed us to discriminate between unique cDNA products and PCR duplicates . Taken together, individual-nucleotide resolution CLIP (iCLIP) enables precise mapping of protein-RNA interactions in intact cells. ArrayExpress Release Date: 2010-09-03 Publication Title: iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution Publication Author List: Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J. Person Roles: submitter Person Last Name: Konig Person First Name: Julian Person Email: jkonig@mrc-lmb.cam.ac.uk Person Phone: 0044 7531 502686 Person Address: Hills Road, CB2 0QH Cambridge, UK Person Affiliation: MRC LMB

ORGANISM(S): Homo sapiens

PROVIDER: GSE25681 | GEO | 2010/12/04

SECONDARY ACCESSION(S): PRJNA135829

REPOSITORIES: GEO

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Project description:Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or a few conditions. We applied direct high-throughput sequencing of cDNAs, complemented with different expression data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including exponential proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at unprecedented resolution. Unlike hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not actively transcribed or rapidly degraded. The combined sequencing and strand-specific array data provided rich information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus refining the existing genome annotation. Sequence trans-reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes. Splicing efficiency was largely coordinated with transcriptional efficiency, and hundreds of introns showed regulated splicing as a function of cellular proliferation or differentiation. ArrayExpress Release Date: 2008-12-03 Publication Title: Dynamic repertoire of a eukaryotic transcriptome surveyed at single nucleotide resolution Publication Author List: Brian Wilhelm, Samuel Marguerat, Stephen Watt, Falk Schubert, Valerie Wood, Ian Goodhead, Christopher J. Penkett, Jane Rogers and Jurg Bahler Person Roles: submitter Person Last Name: Wilhelm Person First Name: Brian Person Mid Initials: Person Email: btw@sanger.ac.uk Person Phone: Person Address: Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK Person Affiliation: Wellcome Trust Sanger Institute Person Roles: investigator Person Last Name: Bahler Person First Name: Jurg Person Mid Initials Person Email: jurg@sanger.ac.uk Person Phone Person Address: Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK Person Affiliation: Wellcome Trust Sanger Institute

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[E-MTAB-341] Individual-nucleotide resolution CLIP using high throughput sequencing of human HeLa cells to enable precise mapping of protein-RNA interactions in intact cells

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