Similar Datasets
Project description:mir-154 in mouse lung development - pull-down RNA
| PRJNA593006 | ENA
Project description:RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)
| PRJNA561512 | ENA
Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual.<br><br>Microarray analysis:<br>The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. <br>Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.
2012-06-30 | E-MEXP-3057 | biostudies-arrayexpress
Project description:RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as ‘incurable’. Here we developed a fluorescent on-bead screening platform: RNA Pull-Down-COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7’s primary target is an mRNA of α-Synuclein, which contributes to the aetiology of Parkinson’s disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-Synuclein. This opens up new therapeutic avenues towards treatment of Parkinson’s disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.
2022-02-17 | PXD025617 | Pride
Project description:To reveal the direct target and molecular mechanisms underlying the effects of dezocine, a pull-down assay and HPLC-MS/MS were performed.l the direct target and molecular mechanisms underlying the effects of dezocine, a pull-down assay and HPLC-MS/MS were performed.
2021-09-09 | PXD022583 | Pride
Project description:In order to investigate the proteins interacting with precusor of miR-276 (pre-miR-276), the RNA-pull down followed by mass spectrometry (MS) were performed in locust terminal oocyte extracts. The RNA oligos labeled with biotin at the 3’ end for pre-miR-276 (62 nt) and pre-miR-67 (69 nt) of C. elegans (negative control) were used. All the bands that were specifically present in the pre-miR-276 group in PAGE were cut and subjected to MS (MS/MS) analysis.
2023-04-26 | PXD041810 |
Project description:The gene body regions of cardiomyocyte-specific genes in cardiomyocytes are hypomethylated. We confirmed that the DNA methylation in gene body regions were demethylated during development. We speculated that the demethylation of gene body regions in cardiomyocyte-specific genes might be associated with active demethylation by Tet oxidation.To explore 5hmC distribution in cells and tissues, we performed 5hmC-specific chemical labeling-mediated pull-down DNA sequencing (hMe-Seal)(Song 2011 Nat genet, PMID:21151123). We found that the 5hmC in gene body regions are associated with demethylation, but not exclusively, and also with transcriptional activity. We concluded that gene body DNA hypomethylation in cardiomyocyte specific genes were mediated by oxidative demethylation.
2016-09-22 | GSE87165 | GEO
Project description:PUF2 RNAi, V5-PUF2 and UBP1-myc pull-downs, all methods and protocols are described in detail in the publication
2018-05-21 | E-MTAB-5617 | biostudies-arrayexpress
Project description:Pull-down of PTP-tagged EIF4E2 from bloodstream-form Trypanosoma brucei, sequence of bound and unbound fractions
2021-08-31 | E-MTAB-10670 | biostudies-arrayexpress
Project description:RNA pull-down assays were performed as the protocol of the PureBinding RNA-Protein pull-down Kit (Geneseed, Guangzhou, China). Specific biotinylated probes that hybridize to target pri-miR-365 were obtained from Ribobio. The probes (200pmol) targeting pri-miR-365 or control probes and RNA samples of chondrocytes were added to the pull-down system. After the mixture of RNA and probes was denaturated at 85°C for 5 minutes, and hybridized at room temperature for 2 hours, 100 µl streptavidin-coated magnetic beads were added to the mixture. Non-specifically bound RNAs were removed by hybridization, capture, and washing. The proteins that interacted with pri-miR-365 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, pri-miR-365-interacting bands were subjected to mass spectrometry (Beijing Genomics Institute, Shenzhen, China). The result was gained to establish a proteomic library.
2022-11-21 | PXD038257 |