biostudies-arrayexpressDomenico MemoliHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16473AKAP1 is highly expressed in glioblastoma and is required for tumor growth. We therefore analyzed the AKAP1 RNA interactome in glioblastoma cells. Native RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) was performed in U87MG cells transiently expressing V5-tagged mouse wild-type AKAP1, as well as deletion mutants lacking either the KH (Δ563–630) or Tudor (Δ709–786) domain, to assess domain-specific RNA binding.biostudies-arrayexpressGrowth Protocol - The human glioblastoma cell line U87MG was cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, L-glutamine, penicillin, streptomycin, and Amphotericin B in a humidified incubater in the presence of 5% CO2.Sample Collection - For each AKAP1-associated RNA immunoprecipitation (RIP) reaction, an anti-V5 antibody was conjugated to sheep anti-mouse IgG–conjugated beads. Protein lysates prepared from U87MG cells transiently transfected with empty vector, wild-type AKAP1, or AKAP1 deletion mutants were incubated with the antibody-conjugated beads. An aliquot of diluted protein lysate was reserved for subsequent RNA extraction as the “input” control. Bead–lysate mixtures were incubated overnight at 4 °C with rotation, followed by four washes with NT2 buffer.Nucleic Acid Extraction - Input samples and washed post-IP reaction beads were resuspended in NT2 buffer and treated with proteinase K to release RNAs from RNA–protein complexes. RNA was then purified using the RNA Clean & Concentrator™-5 Kit (Zymo Research) according to the manufacturer’s instructions.Sequencing - An equimolar pool of libraries at a concentration of 1.2 nM was sequenced on the NovaSeq 6000 platform (Illumina) using 2 × 100 bp paired-end mode.Library Construction - Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina).Sample Treatment - Vectors encoding wild-type or mutant AKAP1 were transiently transfected into U87MG cells using linear polyethylenimine.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - normalization performed using deseq2 default procedureSequence Alignment - we used STAR alignment tool using human h38 as referenceMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RIP-seqHomo sapiensDomenico MemolifalseRNA sequencing–based determination of the RNA interactome of wild-type AKAP1 and its deletion mutants deficient for the KH or Tudor domain.AKAP1 is highly expressed in glioblastoma and is required for tumor growth. We therefore analyzed the AKAP1 RNA interactome in glioblastoma cells. Native RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) was performed in U87MG cells transiently expressing V5-tagged mouse wild-type AKAP1, as well as deletion mutants lacking either the KH (Δ563–630) or Tudor (Δ709–786) domain, to assess domain-specific RNA binding.2026-09-01T00:00:00Z2026-09-01T01:00:38.746Z2025-12-22T18:55:40.225ZE-MTAB-16473ERP187053EFO_0002944EFO_0004170EFO_0003789EFO_0005310EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969