{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Claire Peart"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15274"],"description":["High throughput transcriptomics using the TempO-Seq (TM) platform for dose response modelling of 3 different cell lines treated with multiple compounds as part of a study to generate a toolbox and workflow for non-animal safety assessments. The data generated here was produced as one part of a toolbox of technologies to inform on bioactivity and produced across multiple dates."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Library construction was performed at Bioclavis according to standard protocols. In brief RNA in cell lysate solution was hybridized with the provided detector oligo (DO) pool. TempO-Seq DO pairs were designed to target a 50 nt sequence per gene, with each DO targeting 25 bases (Yeakley et al 2017) The detector pool used was Human Whole genome version 2.1 containing approximately 21000 oligos covering 19300 genes. Subsequently the mix was treated as follows, a nuclease digestion step, a ligation step, followed by heat denaturation. The ligation product was then transferred to an amplification microplate containing PCR mix per well. Ligation products were uniquely labeled during product amplification, with well-specific, “barcoded” primer pairs. Sample amplicons from each well was subsequently pooled into a single sequencing library. The TempO-Seq library was further processed using a PCR clean-up kit. TempO-Seq Human whole genome attenuation panel v 2.1 was used to selectively attenuate highly expressed transcripts","Nucleic Acid Extraction - RNA was prepared according to the standard TempO-Seq protocol (Yeakley et al 2017) which generates a cell lysate without further purification","Sequencing - Sequencing was achieved using a 50 single-end read mode to achieve an average read depth of 200 per gene. Sample demultiplexing was performed using the default Illumina sequencer and bcl2fastq settings see Yeakley et al 2017 A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling","Growth Protocol - The cell line HepG2 (human hepatoblastoma) was obtained from Public Health England European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were cultured in complete Minimal Essential Medium (EMEM) supplemented with 10% foetal bovine serum (FBS), 2 mM L-GlutaMAX, 1% non-essential amino acids (NEAA), 53 U/mL penicillin and 53 µg/mL streptomycin in 75 cm2 cell culture flasks. Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were kept at a confluence below 85% and not maintained in culture more than 4 weeks (8 passages). MCF-7 cells (Human Caucasian breast adenocarcinoma) was obtained from ECACC (Salisbury, UK). Cells were cultured in complete RPMI supplemented with 10% foetal bovine serum (FBS), 2 mM L-GlutaMAX, 53 U/mL penicillin and 53 µg/mL streptomycin. HepaRG cells (Cryopreserved differentiated) were obtained from Caltag Medsystems (UK). HepaRG cells were cultured in Williams E medium supplemented with 2 mM L-glutamine and HPRG670 supplement (Lonza, UK),. HepaRG cells were changed in to serum free medium following the initial 24 hour seeding procedure (Williams E medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and HPRG640 supplement), for six days prior to dosing, with media replenishment every second day. HepG2 and MCF-7 cells were seeded onto 384 well, clear bottom black walled tissue culture plates at a density of 3,000 cell/well and left overnight to attach. HepaRG cells were seeded in collagen coated 384 well, clear bottom black walled tissue culture plates, at a density of 20,000 cells","Sample Treatment - Compound was prepared as a stock solution at 200x higher concentration than the desired top concentration (solvent concentration maintained at 0.5% DMSO). Serial dilutions were performed to generate seven different concentrations of the compound with 0.5% DMSO used as a vehicle control. 5 biological replicates were generated at independent times with the appropriate dose of compound for 24 hours in a humidified atmosphere with 5% CO2 at 37°C.","Sample Collection - Following treatment, cell media was aspirated and cells washed with PBS. After removal of all residual PBS, 2X TempO-Seq Enhanced lysis buffer (BioSpyder Technologies, proprietary kit) was diluted to 1X with PBS and added at a volume of 1µL per 1000 cells with a minimum of 10 µL per well and incubated for 10 min at room temperature. Following lysis, the samples were frozen at -80°C prior to sequencing"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Sequences were aligned to Biospyder probe manifest Homo sapiens whole genome ver 1.1 using the STAR alignment programme Dobin et al Bioinformatics, Volume 29, Issue 1, January 2013, Pages 15-21","Data Transformation - Data was assessed through standard QC pipelines to ensure sufficient read depth, replicate correlation, and mapping %. Downstream analysis followed either DeSEQ2 normalisation or use of Bifrost dose response modelling (Reynolds et al 2020 A Bayesian approach for inferring global points of departure from transcriptomics data)"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 2500"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_title":["An Advancement in Developmental and Reproductive Toxicity (DART) Risk Assessment: Evaluation of a Bioactivity and Exposure-Based NAM Toolbox"],"pubmed_authors":["Iris Müller, Ashraf Abdelkhaliq, Paul Carmichael, Matthew Dent, Marleen Feliksik, Luke Flatt, Jade Houghton, Jose Horcas-Nieto, Amer Jamalpoor, Predrag Kukic, Sophie Malcomber, Beate Nicol , Gopal Pawar, Claire Peart, Katarzyna Przybylak, Magdalena Sawicka, Katy Wilson, Kathryn Wolton","Claire Peart"],"additional_accession":[]},"is_claimable":false,"name":"An Advancement in Developmental and Reproductive Toxicity (DART) Risk Assessment: Evaluation of a Bioactivity and Exposure-Based NAM Toolbox","description":"High throughput transcriptomics using the TempO-Seq (TM) platform for dose response modelling of 3 different cell lines treated with multiple compounds as part of a study to generate a toolbox and workflow for non-animal safety assessments. The data generated here was produced as one part of a toolbox of technologies to inform on bioactivity and produced across multiple dates.","dates":{"release":"2025-09-15T00:00:00Z","modification":"2025-09-15T01:02:31.775Z","creation":"2025-06-26T12:04:45.309Z"},"accession":"E-MTAB-15274","cross_references":{"ENA":["ERP177330"],"Biostudies":["E-MTAB-14069"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184","EFO_0003969"],"doi":["10.3389/ftox.2025.1602065"]}}