ICLIP of GFP fusion proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Homo sapiens
SUBMITTER: Christian Hauer
PROVIDER: E-MTAB-4215 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37°C. For iCLIP, HeLa cells expressing eIF4A3-GFP or PTB-GFP were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries for eIF4A3 and PTB were sequenced with 50 bp on an Illumina HiSeq instrument.
2015-08-11 | E-MTAB-2599 | biostudies-arrayexpress
Project description:2-3 month old female C57Bl/6J mice were intravenously infected with 2x10^6 focus forming units of lymphocytic choriomeningitis virus strain Clone-13. Whole liver tissue was harvested in triplicates from uninfected control mice, uninfected control 123d old mice, and from infected mice at 2, 8, 30, 60, and 123 days post infection. Samples were subsequently analyzed on a HiSeq 2000 instrument using a 50 bp SE RNAseq protocol.
2019-12-05 | GSE118703 | GEO
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
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
2016-06-23 | E-MTAB-3612 | biostudies-arrayexpress
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
Project description:This experiments was performed in HeLa cells according to the iCLIP protocol with the following modifications: no antiRNase was used and the concentration of RNase I was 0.5 U/ml. In iCLIP4, the dephosphorylation step was omitted from the standard protocol. The rest of the protocol was identical to the previously published iCLIP protocol )Huppertz I, Attig J, D'Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Knig J, Ule J: iCLIP: protein-RNA interactions at nucleotide resolution. Methods 2014, 65:274-287).
2016-11-24 | E-MTAB-5026 | biostudies-arrayexpress
Project description:iCLIP of HeLa cells with anti-TFIP11 antibody to investigate RNA binding locations of TFIP11 as part of a larger study on the function of TFIP11.
2021-09-22 | GSE158589 | GEO
Project description:Purpose: The aim of this study was to determine the DNA methylation state of wildtype female mouse embryonic fibroblasts with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Enhanced Reduced Representation Bisulfite Libraries (eRRBS) were produced as previously descirbed (Akalin et al. 2012). Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads with dark cycle parameters (Boyle et al. 2012). Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
2016-05-18 | GSE66524 | GEO
Project description:The modified iCLIP protocol is based on the previously described protocol (Huppertz et al., 2014) with modifications that enable the definition of readthrough cDNAs. HeLa cells were crosslinked with 0.15mJ/cm2 254nm UV light. Protein G Dynabeads were used for immunoprecipitations (IP). For each IP, 100 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCL pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and incubated with the anti-eIF4A3 antibody or polyclonal mAb BB7 serum anti-PTBP1. To prepare the cell lysate, the cells were lysed with 1 mL iCLIP lysis buffer (final concentration 2mg/mL), sonicated (Bioruptor, 5x5 sec on/off), incubated with RNase I (1-2 x10-3 U/mL for eIF4A3) at 37C for 3 min, and centrifuged. After the pellet had dissolved, the mixture was diluted with H2O to 1000 L and an additional centrifugation was performed. The supernatant of the iCLIP lysis buffer (1 mL) and the MSB lysis buffer (1 ml) were combined and added to the antibody-coupled beads, which were then rotated at 4C for 2 h (final urea concentration 175 mM). The beads were then washed with high-salt washing buffer (50mM Tris-HCL pH 7.4, 1M NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). After the first round of washes, the library was split into 10%, which were radioactively labelled (according to the basic iCLIP protocol), and 90%, which proceed through 3 adapter addition, an additional phosphorylation (0.2 l PNK, 0.4 l cold ATP (1mM), 0.4 l 10x PNK buffer, 3 l water) and a 5 marker ligation (6 l water, 5 l 4X ligation buffer, 2 l RNA ligase, 1 l RNasin, 2 l 5 marker (100 M), 4 l PEG400). The sequence of the 5 marker is CAGUCCGACGAUC, which corresponds to the Illumina short RNA 5 Adapter (RA5), part #15013205; this sequence is not complementary to the primers used for amplification of iCLIP cDNA libraries (Huppertz et al., 2014). We then produced sequence reads of 120 nt using the Illumina HiSeq platform for eIF4A3 iCLIP. The rest of the protocol was carried out as previously described (Huppertz et al., 2014). Huppertz, I., Attig, J., D'Ambrogio, A., Easton, L.E., Sibley, C.R., Sugimoto, Y., Tajnik, M., Konig, J., and Ule, J. (2014). iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287.
2016-11-24 | E-MTAB-4000 | biostudies-arrayexpress
Project description:Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ Xist∆A/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data.
2016-05-18 | GSE66523 | GEO