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Next Generation Sequencing Facilitates Quantitative Analysis of genome-wide binding sites of hnRNP U in BNL CL.2 cells.

ABSTRACT: Purpose:The aim of this study is to evaluate the characteristics of hnRNP U binding site. Methods: The chromatin of BNL CL.2 cells was immunoprecipitated with anti-hnRNP U(Abcam) and normal rabbit immunoglobulin G (IgG). The immunoprecipitated DNA was then subjected to various previously constructed sequencing libraries. The libraries were sequenced using the Illumina Hiseq2500. All reads were aligned to the mm9 genome browser using the bowtie with default parameters. Results: A total number of 12249 peaks that specifically binded by hnRNP U was obtained. Conclusions: Our study represents the first detailed analysis of genome-wide binding sites of hnRNP U generated by ChIP-seq technology.

ORGANISM(S): Mus musculus

PROVIDER: GSE75335 | GEO | 2015/11/25

SECONDARY ACCESSION(S): PRJNA303969

REPOSITORIES: GEO

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Next Generation Sequencing Facilitates Quantitative Analysis of genome-wide binding sites of hnRNP U in BNL CL.2 cells.

Project description:Purpose:The aim of this study is to evaluate the characteristics of hnRNP U binding site. Methods: The chromatin of BNL CL.2 cells was immunoprecipitated with anti-hnRNP U(Abcam) and normal rabbit immunoglobulin G (IgG). The immunoprecipitated DNA was then subjected to various previously constructed sequencing libraries. The libraries were sequenced using the Illumina Hiseq2500. All reads were aligned to the mm9 genome browser using the bowtie with default parameters. Results: A total number of 12249 peaks that specifically binded by hnRNP U was obtained. Conclusions: Our study represents the first detailed analysis of genome-wide binding sites of hnRNP U generated by ChIP-seq technology. hnRNP U binding profiling in Mus musculus fetal liver cell line BNL CL.2 cells, and the IgG antibody was used as a negative control

2015-11-25 | E-GEOD-75335 | biostudies-arrayexpress

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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.

2018-06-27 | E-MTAB-6221 | biostudies-arrayexpress

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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

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hnRNP A1 iCLIP

Project description:hnRNP A1 iCLIP

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The novel role of hnRNP UL1 in human cell nucleoli

Project description:hnRNPUL1 plays an important role in the cell nuclei, where it is recruited to DNA damage sites and is involved in the repair of DNA double-strand breaks. Furthermore, this protein is known as a transcriptional repressor of RNA polymerase II genes. In the present study, we have shown that hnRNP UL1 is also localized in the nucleoli. Revealing its function, we figured out that hnRNP UL1 stimulates ribosomal DNA (rDNA) gene transcription. We have performed high-throughput sequencing of libraries prepared from nucleolar and cytoplasmic-nuclear RNA fractions to identify differentially expressed ribosomal RNAs in wild-type and HNRNPUL1 knockout HEK293T cells.

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