biostudies-arrayexpressVolker BöhmHomo sapiensSTAR read aligner (version 2.7.10b)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15521Nonsense-mediated decay (NMD) is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. UPF1 is the central NMD factor required for PTC recognition and subsequent recruitment of the executing factors SMG5 and SMG6. To study the impact of UPF1, SMG5 or SMG6 protein depletion on the transcriptome, we established dTAGV-1-inducible degron systems in the human embryonic kidney cell line HEK293 (Flp-In-T-REx-293) by tagging the respective protein at the N-terminus with an Myc-FKBP-tag (FKBP = FKBP12-F36V). FKBP-tagged SMG5 and SMG6 cell lines were treated for 72h with 60 pmol siRNAs targeting the respective mRNA, whereas FKBP-tagged UPF1 and the parental cell line was treated similarly with control Luciferase siRNA. Protein degradation of UPF1, SMG5 and SMG6 was induced with 0.25 µM dTAGV-1 for 24h (48h after RNAi), whereas control cells were treated with DMSO.biostudies-arrayexpressLibrary 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).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 Treatment - 2.8x10^5 cells were seeded in 6-well plates and reverse transfected with 2.5 µl Lipofectamine RNAiMAX (Invitrogen; Cat# 13778150) and 60 pmol of the respective siRNAs (Luciferase- or SMG5/SMG6-targeting) according to the manufacturer’s protocol. Medium was changed the next day and 48 hours after knockdown, protein depletion was induced with 0.25 µM dTAGV-1 (Tocris Bioscience; Cat# 6914) for 24 hours, same volumes of DMSO served as controls. Thus, the cells were harvested 3 days after the initial knockdown.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).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData 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).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensNiels GehringVolker BöhmfalseRNA-Seq of UPF1, SMG5 or SMG6 depletion in human embryonic kidney cell line HEK293 via the dTAG degron system and siRNA-mediated knockdownsNonsense-mediated decay (NMD) is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. UPF1 is the central NMD factor required for PTC recognition and subsequent recruitment of the executing factors SMG5 and SMG6. To study the impact of UPF1, SMG5 or SMG6 protein depletion on the transcriptome, we established dTAGV-1-inducible degron systems in the human embryonic kidney cell line HEK293 (Flp-In-T-REx-293) by tagging the respective protein at the N-terminus with an Myc-FKBP-tag (FKBP = FKBP12-F36V). FKBP-tagged SMG5 and SMG6 cell lines were treated for 72h with 60 pmol siRNAs targeting the respective mRNA, whereas FKBP-tagged UPF1 and the parental cell line was treated similarly with control Luciferase siRNA. Protein degradation of UPF1, SMG5 and SMG6 was induced with 0.25 µM dTAGV-1 for 24h (48h after RNAi), whereas control cells were treated with DMSO.2026-01-30T00:00:00Z2026-01-31T02:02:37.509Z2025-09-02T10:51:23.44ZE-MTAB-15521ERP179476EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969