biostudies-arrayexpressTamara DanilyukHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15401We aimed to compare the baseline expression of liver function and metabolism-related genes across the three liver models. Liver in vitro models are commonly used to study liver biology, disease mechanisms, and drug metabolism. Understanding their baseline gene expression profiles is crucial for interpreting experimental results and selecting the most appropriate model for specific research questions. Experimental workflow Cell Culture: Culture HepG2, PHHs, and hiPSCs-derived HLCs under standard, untreated conditions. Cell lysis: Collect RNA lysates using BioSpyder 2x enhanced lysis buffer from each cell type. Sequencing: Ship lysates to BioClavis, 201 Dumbarton Rd, Glasgow G81 4XJ, United Kingdom and sequence using TempoSeq targeted sequencing technology: https://www.bioclavis.co.uk/temposeq Data Analysis: Perform quality control of the sequencing results, normalize the data and compare normalized gene expression profiles, focusing on liver function and metabolic genes across the three models.biostudies-arrayexpressLibrary Construction - Cell lysates were shipped and processed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology, using the human whole-transcriptome probeset version 2.0 according to the manufacturer's standard procedure. Each Detector Oligo (DO) contains a region complementary to its specific mRNA target and a universal primer binding site shared across all targets. DOs hybridize adjacently on the RNA template, enabling their ligation into a single molecule. These ligated oligos are then PCR-amplified in a single-plex reaction using a universal primer pair, which includes both the sequencing adaptors and a unique sample barcode. The barcode sequences are positioned to flank the target region and are integrated into the standard Illumina adaptors. This facilitates dual-index sequencing for accurate sample identification and de-multiplexing. All barcoded PCR products are pooled into a single sequencing library. Sequencing reads are demultiplexed by the standard Illumina software using the sample-specific barcodes, resulting in individual FASTQ files for each sample.Nucleic Acid Extraction - Cell lysates were shipped and processed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology, using the human whole-transcriptome probeset version 2.0 according to the manufacturer's standard procedure. Each Detector Oligo (DO) contains a region complementary to its specific mRNA target and a universal primer binding site shared across all targets. DOs hybridize adjacently on the RNA template, enabling their ligation into a single molecule. These ligated oligos are then PCR-amplified in a single-plex reaction using a universal primer pair, which includes both the sequencing adaptors and a unique sample barcode. The barcode sequences are positioned to flank the target region and are integrated into the standard Illumina adaptors. This facilitates dual-index sequencing for accurate sample identification and de-multiplexing. All barcoded PCR products are pooled into a single sequencing library. Sequencing reads are demultiplexed by the standard Illumina software using the sample-specific barcodes, resulting in individual FASTQ files for each sample.Sequencing - Cell lysates were shipped and processed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology, using the human whole-transcriptome probeset version 2.0 according to the manufacturer's standard procedure. Each Detector Oligo (DO) contains a region complementary to its specific mRNA target and a universal primer binding site shared across all targets. DOs hybridize adjacently on the RNA template, enabling their ligation into a single molecule. These ligated oligos are then PCR-amplified in a single-plex reaction using a universal primer pair, which includes both the sequencing adaptors and a unique sample barcode. The barcode sequences are positioned to flank the target region and are integrated into the standard Illumina adaptors. This facilitates dual-index sequencing for accurate sample identification and de-multiplexing. All barcoded PCR products are pooled into a single sequencing library. Sequencing reads are demultiplexed by the standard Illumina software using the sample-specific barcodes, resulting in individual FASTQ files for each sample.Sample Collection - All cell cultures were done in 96 well plates and washed once with DPBS without calcium and magnesium chloride (Sigma-Aldrich, #D8537). Next, 50 µL lysis buffer (1:1 DPBS and 2x TempO‐Seq lysis buffer, BioSpyder) was added to the cells for 10 minutes at 37°C. Plates were immediately sealed with an aluminum silver seal (Greiner Bio-One, Cat #676090) and stored at -80°C until shipped to BioClavis, UK.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - A count table of the targeted RNA sequencing reads was provided by BioClavis, containing 27 samples (3 cell types, 9 replicates) with 22533 measured probes. Samples with less than 500,000 counts were removed as these were outliers from the average of 3,000,000 reads per sample. Samples were normalised using the DESeq2 package (version 1.36.0) in R (version 4.1.0 or newer) by applying a counts per million (CPM) normalisation (Love et al., 2014; R Core Team, 2022). Low expressed probes were removed from the raw expression matrix according to the relevance filter of the RNA-seq R-ODAF pipeline (Verheijen et al., 2022). Next, multiple probes for the same gene were combined by taking the sum of the probe counts. CPM normalisation was applied again by dividing raw counts by the sizefactors of each sample in the filtered raw expression matrix. Normalized counts of relevant genes were compared across three liver models.UnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2500RNA-seq of coding RNAHomo sapiensMathematical modelling reveals compound-specific stress pathway activity.Elsje J. Burgers1, Tamara Y. Danilyuk1, Raju P. Sharma1, Nadine Renner2, Andreas Verlohner3, Nicole Rocker4, Philipp Ternes4, Lukas S. Wijaya1, Marcel Leist5, Peter Bouwman1, Franziska M. Zickgraf3, Stefan Schildknecht2, Bob van de Water1, Joost B. Beltman1*Tamara DanilyukfalseTargeted RNA-seq (TempO-Seq) of human hepatocellular carcinoma cell line HepG2, primary human hepatocytes (PHHs) and human induced pluripotent stem cell derived hepatocyte like cells (HLCs) in untreated conditionWe aimed to compare the baseline expression of liver function and metabolism-related genes across the three liver models. Liver in vitro models are commonly used to study liver biology, disease mechanisms, and drug metabolism. Understanding their baseline gene expression profiles is crucial for interpreting experimental results and selecting the most appropriate model for specific research questions. Experimental workflow Cell Culture: Culture HepG2, PHHs, and hiPSCs-derived HLCs under standard, untreated conditions. Cell lysis: Collect RNA lysates using BioSpyder 2x enhanced lysis buffer from each cell type. Sequencing: Ship lysates to BioClavis, 201 Dumbarton Rd, Glasgow G81 4XJ, United Kingdom and sequence using TempoSeq targeted sequencing technology: https://www.bioclavis.co.uk/temposeq Data Analysis: Perform quality control of the sequencing results, normalize the data and compare normalized gene expression profiles, focusing on liver function and metabolic genes across the three models.2025-08-09T00:00:00Z2025-07-25T11:54:18.801Z2025-07-25T11:54:18.801ZE-MTAB-15401ERP177267EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184