title: GSM2552915: iPAR-CLIP_L001; Homo sapiens; RIP-Seq
library_construction_protocol: The PAR-iCLIP was carried out according to the protocol described by: 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. Briefly, MCF-7 cells (1 x 107) were seeded 48 h before they were incubated with 100 µM 4-thiouridine (4-SU) for 4 h. For cross-linking purposes, cells were treated with UV light (365 nm, 4000 mJ/cm²) directly before lysis. The PAR-iCLIP was carried out according to the protocol described by: 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. hnRNPA1, and the cross-linked RNA, was immunoprecipitated using 6 µg anti-hnRNPA1 antibody and 100 µL Dynabeads® protein G (Thermo Fisher Scientific). The RNA was radiolabeled and the hnRNPA1-RNA complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. RNA was isolated from the membranes by digestion of the protein with proteinase K (Thermo Fisher Scientific) and reverse transcribed. The resulting cDNA was gel purified and amplified with primers containing the adapter sequences for subsequent next generation sequencing (NGS), which was carried out on a NextSeq500 platform (Illumina, San Diego, USA).
attributes.tag: GEO Accession
attributes.value: GSM2552915