{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Volker Böhm"],"organism":["Homo sapiens"],"software":["STAR read aligner (version 2.7.10b)"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16399"],"description":["Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7.  UPF1 phosphorylation is mediated by the kinase SMG1, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here, we established SMG8- and SMG9-depleted human osteosarcoma U2OS cells (Flp-In-T-REx-U2OS). With these cell lines we wanted to explore the regulatory role of SMG8 and SMG9 for NMD execution. Furthermore, we tested the transcriptomic changes upon treatment of cells with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h. As controls, the U2OS (WT or KO) cells were treated with DMSO."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - ERCC RNA Spike-In Mix 1 (Thermo Fisher Scientific, Cat# 4456740) was added to the total RNA sample before library preparation. Libraries was prepared using the Stranded mRNA Preparation Kit (Illumina). Library preparation started with 500ng total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second strand cDNA synthesis. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (12 PCR cycles) to create the final cDNA library. After validation (TapeStation, Agilent Technologies) and quantification (Qubit, Thermo Fisher Scientific) individual libraries were pooled.","Growth Protocol - All cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122).","Sample Treatment - 2.5x10^5 cells were seeded in 6-well plates and treated 2 days later for 24 hours with DMSO as control, 0.1 µM or 1 µM SMG1i. Thus, the cells were harvested 3 days after the seeding.","Nucleic Acid Extraction - Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions.","Sequencing - The library pools were quantified using the Collibri Library Quantification Kit (Thermo Fisher Scientific) and the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Libraries were subsequently sequenced on an Illumina NovaSeq 6000 instrument using a 2x100 bp sequencing protocol and aiming for 50 million clusters per sample.","Sample Collection - Cells were harvested and lysed by adding 1 ml of in-house prepared TRI reagent to each well (prepared following DOI: 10.1371/journal.pbio.3000107)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Reads were aligned against the human genome (GRCh38, GENCODE release 42 transcript annotations supplemented with SIRVomeERCCome annotations from Lexogen; obtained from https://www.lexogen.com/sirvs/download/) using the STAR read aligner (version 2.7.10b, https://github.com/alexdobin/STAR)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Niels Gehring","Volker Böhm"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of SMG1-SMG8-SMG9 complex alterations in human osteosarcoma cell line U2OS","description":"Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7.  UPF1 phosphorylation is mediated by the kinase SMG1, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here, we established SMG8- and SMG9-depleted human osteosarcoma U2OS cells (Flp-In-T-REx-U2OS). With these cell lines we wanted to explore the regulatory role of SMG8 and SMG9 for NMD execution. Furthermore, we tested the transcriptomic changes upon treatment of cells with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h. As controls, the U2OS (WT or KO) cells were treated with DMSO.","dates":{"release":"2026-02-13T00:00:00Z","modification":"2026-05-26T10:11:55.755Z","creation":"2025-12-17T22:32:53.67Z"},"accession":"E-MTAB-16399","cross_references":{"ENA":["ERP186721"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"]}}