biostudies-arrayexpressClaire PeartHomo sapiensSTARbcl2fastqhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15258High 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.biostudies-arrayexpressLibrary 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 transcriptsNucleic Acid Extraction - RNA was prepared according to the standard TempO-Seq protocol (Yeakley et al 2017) which generates a cell lysate without further purificationSequencing - 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 profilingGrowth 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 cellsSample 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 sequencingOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence 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 15-21Data 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)MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2500An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions. The toolbox includes physiologically based kinetic (PBK) models to estimate systemic Cmax levels in humans, and 3 bioactivity platforms, comprising high-throughput transcriptomics, a cell stress panel, and in vitro pharmacological profiling, from which points of departure are estimated. A Bayesian model was developed to quantify the uncertainty in the Cmax estimates depending on how the PBK models were parameterized. The feasibility of the evaluation approach was tested using 24 exposure scenarios from 10 chemicals, some of which would be considered high risk from a consumer goods perspective (eg, drugs that are systemically bioactive) and some low risk (eg, existing food or cosmetic ingredients). Using novel protectiveness and utility metrics, it was shown that up to 69% (9/13) of the low risk scenarios could be identified as such using the toolbox, whilst being protective against all (5/5) the high-risk ones. The results demonstrated how robust safety decisions could be made without using animal data. This work will enable a full evaluation to assess how protective and useful the toolbox and workflow are across a broader range of chemical-exposure scenarios.RNA-seq of total RNAHomo sapiensAre Non-animal Systemic Safety Assessments Protective? A Toolbox and WorkflowAlistair M. Middleton, Joe Reynolds, Sophie Cable, Maria Teresa Baltazar, Hequn Li , Samantha Bevan, Paul L. Carmichael, Matthew Philip Dent, Sarah Hatherell, Jade Houghton, Predrag Kukic, Mark Liddell, Sophie Malcomber, Beate Nicol, Benjamin Park, Hiral Patel, Sharon Scott, Chris Sparham, Paul Walker ,and Andrew WhiteClaire PeartAlistair MiddletonfalseAre Non-animal Systemic Safety Assessments Protective: A Toolbox and WorkflowHigh 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.2025-09-15T00:00:00Z2025-09-15T01:02:31.788Z2025-06-26T11:27:33.898ZE-MTAB-1525835822611ERP177323EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_000396910.1093/toxsci/kfac068