{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Simon Henriet"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15845"],"description":["The 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."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample 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 precipitated","Sequencing - 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."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data 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."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Bio-Rad Mini-PROTEAN electrophoresis system and wet transfer cassettes,","NextSeq 500","Bio-Link Crosslinker BLX-E254"],"study_type":["CLIP-seq"],"species":["Homo sapiens"],"pubmed_authors":["Simon Henriet"],"additional_accession":[]},"is_claimable":false,"name":"Binding sites of Fritillaria borealis U2AF2 expressed in HEK293T cells","description":"The 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.","dates":{"release":"2026-06-11T00:00:00Z","modification":"2026-06-11T07:15:10.178Z","creation":"2025-10-23T23:43:40.113Z"},"accession":"E-MTAB-15845","cross_references":{"ENA":["ERP182852"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003143","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}