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Individual-nucleotide resolution CLIP and high-throughput sequencing to map hnRNP L-RNA interactions in HeLa cells

ABSTRACT: Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments.

ORGANISM(S): Homo sapiens

PROVIDER: GSE37560 | GEO | 2014/02/13

SECONDARY ACCESSION(S): PRJNA162423

REPOSITORIES: GEO

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Individual-nucleotide resolution CLIP and high-throughput sequencing to map hnRNP L-RNA interactions in HeLa cells

Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells

2014-02-13 | E-GEOD-37560 | biostudies-arrayexpress

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hnRNP A1 iCLIP

Project description:hnRNP A1 iCLIP

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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

Project description: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

2010-12-04 | GSE25681 | GEO

hnRNP A1 iCLIP

Project description:This experiment identifies hnRNP A1 binding sites transcriptome-wide in Hela cells. HeLa cells with inducible expression of T7-tagged hnRNP A1 were grown to approximately 90% confluence and then subject to iCLIP analysis (following the protocol from Huppertz et al. 2014 (iCLIP: protein-RNA interactions at nucleotide resolution)). The iCLIP library was sequenced using Illumina's HighSeq 1500

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Mapping hnRNP H binding sites on the RON wildtype minigene via iCLIP.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene, we performed hnRNP H iCLIP with RON wildtype minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two replicates of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina HiSeq2000 (75-nt single-end reads).

2018-06-27 | E-MTAB-6220 | biostudies-arrayexpress

Mapping hnRNP H binding sites on RON wildtype minigene and RON mutant minigenes via iCLIP.

Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. We identified the RNA binding protein hnRNP H as an important regulator of RON exon 11 splicing. To map hnRNP H binding sites on the RON minigene with either wildtype or hnRNP H binding site mutant background, we performed hnRNP H iCLIP with RON wildtype or mutant minigene transfected HEK293T cells. iCLIP was performed according to a previously published protocol (PMID: 26463384). The iCLIP libraries were made from two (G331C and G348C) or three replicates (wildtype and G305A) of HEK293T cells at 24 h after RON minigene transfection. The cells were irradiated with 150 mJ/cm2 UV light at 254 nm. For the immunoprecipitation step, we used 7.5 µg of a polyclonal rabbit anti-HNRNPH antibody from Abcam (AB10374). RNase digestion was performed by adding 10 µl of 1/100 diluted RNase I (Ambion) to each sample. We performed the sequencing on an Illumina MiSeq or NextSeq500 with 75-nt single-end reads.

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