biostudies-arrayexpressSimon HenrietHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15845The goal of the experiment is to reveal RNA binding preferences of Fritillaria borealis U2AF2 paralogues, using CLIP-seq. Coding sequences of U2AF2alpha, U2AF2beta and Env negative control were fused to a C-terminal V5 tag. Protein expression constructs were transfected in mammalian cell culture. After cross-linking, RNA-protein complexes were recovered with anti-V5 magnetic beads.biostudies-arrayexpressSample Collection - Two days post-transfection, cells were washed with PBS and irradiated on ice with 254 nm light (150 mJ). Cells were resuspended in lysis buffer supplemented with 1 mM PMSF and protease inhibitors (cOmplete-EDTA, Roche), before sonication on ice twice with 10-second pulses. Transcripts were fragmented for 3 minutes at 37 °C in the presence of 1 U/mL TURBO DNase and 20-1000 U/mL RNase I (Ambion). Insoluble material was pelleted by 10 minutes of centrifugation at 22000 g at 4 °C, and supernatant was added to Anti-V5 magnetic beads (Sigma, equivalent to 70 µL suspension), prewashed in lysis buffer. After binding 150 minutes on a wheel at 4 °C, RNPs were washed twice with ice-cold High-salt wash buffer and three times with ice-cold PNK buffer.Nucleic Acid Extraction - Purified RNPs were radiolabelled at 5'end, resolved on Bis-Tricine 8%PAGE and transferred on nitrocellulose. Size-selected RNPs were eluted, digested with proteinase K-SDS, RNA was extracted with Phenol-Chloroform and precipitatedSequencing - pools of barcoded libraries were sequenced on NextSeq HIGH 300 cycles at the Norwegian Sequencing Centre (Oslo, Norway), without index reads.Library Construction - Libraries were prepared following the iCLiP2 protocol (Buchbender et al., Methods 2020). Individual libraries were barcoded by 5'ligation of a DNA adapter to the cDNA, that includes a UMI.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - We used Pureclip trained on chromosomes 1-4 to detect U2AF2 crosslinks, using Env-associated crosslinks as a background control. Crosslink sites shared between U2AF2alpha and U2AF2beta were substracted to produce a collection of unique peaks.Sequence Alignment - Barcoded Q10 reads over 15 bp were demultiplexed, adapters were trimmed and PCR duplicates removed. Reads were mapped on the human genome assembly hg38 with Bowtie2 (--very-sensitive-local). See Busche et al., Methods 2019 for detailed procedures.UnknownTranscriptomicsGenomicsProteomicsBio-Rad Mini-PROTEAN electrophoresis system and wet transfer cassettes,NextSeq 500Bio-Link Crosslinker BLX-E254CLIP-seqHomo sapiensSimon HenrietfalseBinding sites of Fritillaria borealis U2AF2 expressed in HEK293T cellsThe goal of the experiment is to reveal RNA binding preferences of Fritillaria borealis U2AF2 paralogues, using CLIP-seq. Coding sequences of U2AF2alpha, U2AF2beta and Env negative control were fused to a C-terminal V5 tag. Protein expression constructs were transfected in mammalian cell culture. After cross-linking, RNA-protein complexes were recovered with anti-V5 magnetic beads.2026-06-11T00:00:00Z2026-06-11T07:15:10.178Z2025-10-23T23:43:40.113ZE-MTAB-15845ERP182852EFO_0002944EFO_0004170EFO_0003143EFO_0004917EFO_0005518EFO_0003816EFO_0004184