{"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-15870"],"description":["The aim of the experiment is to reveal splicing isoforms induced by transient expression of Fritillaria borealis U2AF in HEK293T cells. Cells were transfected with different combinations of U2AF and U2AF2 subunits paralogues. A cycloheximide treatment was applied to inhibit nonsense-mediated decay and to stabilize transcripts produced by aberrant splicing. After cDNA conversion, target isoforms were amplified and sequenced."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - After removing growth media, cell monolayers were resuspended in PBS. Cells were centrifuged 1 minute at 100g, and the supernatant was removed prior to RNA extraction.","Growth Protocol - Cells were transfected with expression vectors for either Fritillaria borealis subunits U2AF1 and U2AF2 or beta-galactosidase, then kept during 2 days in culture at 37C in DMEM.","Sample Treatment - Cell cultures were treated with 15 mM Cycloheximide during 8 hours at 37C prior to sample collection. Control samples were treated with DMSO.","Sequencing - Pools of barcoded libraries were sequenced on a PromethION flowcell. Basecalling was carried out during sequencing with the high accuracy model.","Nucleic Acid Extraction - Total RNA was extracted with Trizol reagent following the manufacturer's protocol and treated with DNase.","Library Construction - Total RNA was annealed to a mixture of random primers and Oligo-dT and reverse-transcribed. We used 20 cycles of PCR to amplify cDNA in inidiviual reactions carried out with gene-specific primers for ALG13, eIF1B, FMRI and HSP90B1. The PCR reactions corresponding to the same transfection were processed for native barcoding following the protocol of Oxford Nanopore Technology and pooled together."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - We used TALON to annotate isoforms. Isoform expression was normalized on library size.","Sequence Alignment - Selected Q9 basecalled reads longer than 500 bp were trimmed with porechop and aligned with minimap2 to a gene database that includes only the sequences targeted by amplicon primers."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["PromethION"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Simon Henriet"],"additional_accession":[]},"is_claimable":false,"name":"Amplicon cDNA sequencing in cells expressing Fritillaria borealis U2AF","description":"The aim of the experiment is to reveal splicing isoforms induced by transient expression of Fritillaria borealis U2AF in HEK293T cells. Cells were transfected with different combinations of U2AF and U2AF2 subunits paralogues. A cycloheximide treatment was applied to inhibit nonsense-mediated decay and to stabilize transcripts produced by aberrant splicing. After cDNA conversion, target isoforms were amplified and sequenced.","dates":{"release":"2026-06-11T00:00:00Z","modification":"2026-06-11T07:14:34.528Z","creation":"2025-11-13T16:23:23.906Z"},"accession":"E-MTAB-15870","cross_references":{"ENA":["ERP183099"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"]}}