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ICLIP analysis of RNA binding sites of Stau2 protein in mouse brain cells

ABSTRACT: iCLIP analysis of RNA binding sites of Stau2 protein in mouse brain cells

PROVIDER: PRJEB20555 | ENA |

REPOSITORIES: ENA

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iCLIP analysis of RNA binding sites of Stau2 protein in mouse brain cells

Project description:Stau2 iCLIP of mouse brain was performed to identify RNA binding sites of Stau2 protein in mouse brain cells. The experiment was performed in triplicate, and each of the replicates was split into two separate halves at the cDNA stage, which together led to 6 separate datasets. The iCLIP protocol includes the following steps; Embryonic day 18 whole mouse brain was dissociated and irradiated with UV-C light, and the cells were then lysed using a buffer containing detergents. The RNAs were partially digested, and Stau2 and the cross-linked RNA fragments were immunoprecipitated using anti-Stau2 antibody. A DNA adaptor was then ligated to the RNA fragments and the cross-linked RNAs were further purified by SDS-PAGE and nitrocellulose membrane transfer. The RNAs were extracted from the membrane by proteinase K treatment, and converted into a high-throughput DNA sequencing compatible library by reverse transcription and PCR. For further details, see the methods of the associated manuscript. Note that the sequence reads start with (3 nucleotides of unique molecular identifiers) + (4 nucleotides of experimental barcode) + (2 nucleotides of unique molecular identifiers) followed by the sequence of the cross-linked RNA fragments.

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