biostudies-arrayexpressTamara DanilyukHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15398We wanted to investigate the transcriptional effect of Nitrofurantoin, a known drug induced liver injury (DILI)-compound, on hiPSC-derived HLCs. The goal was to identify which srress response pathways and biological processes are activated or altered in HLCs following Nitrofurantoin exposure. The compound is known to cause hepatotoxicity through mechanisms of oxidative stress, mitochondrial dysfunction, but the precise cellular responses in human liver models remain not fully understood.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.Sample Collection - After 8 and 24 hours post-exposure, cells across all conditions were 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.Growth Protocol - hiPSC-derived HLCs were generated following a 40-day differentiation protocol. hiPSCs were seeded at 8,421 cells per well in 96-well plates (Greiner Bio-One, Cat #655161). After 24 hours, the medium was replaced with mTESR1 lacking 1x RevitaCell Supplement. Differentiation was initiated 48–72 hours later, once cells reached 70% confluency, using liver differentiation medium (LDM) supplemented with defined cytokines. From day 0 to 10, the medium was changed every other day and contained 0.6% DMSO. Starting on day 4, 5 µg/mL Doxycycline (Selleckchem, Cat#S5159) was added to induce HC3X expression. On day 12, DMSO was raised to 2%, and both non-essential and essential amino acids (Gibco, Cat#11140050 and Cat#11130051) were added to create LDM-AA. From day 14 to 50, the medium was further supplemented with 20 g/L glycine (LDM-AAGLY), and DMSO was removed. By day 50, cells were ready for exposure experiments. All growth factors were sourced from PeproTech.Sample Treatment - Prior to exposure, 500x stock solutions of Nitrofurantoin (Merck, N7878) were prepared in DMSO. On day 50, HLCs were exposed by adding 50 µL of LDM-AAGly medium containing 4.5 µg/mL doxycycline and 20 ng/mL HGF to wells already containing 50 µL of differentiation medium. All exposures were performed in triplicate, with both biological and technical replicates, using independent compound stock preparations.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.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 induced pluripotent stem cell (hiPSC)-derived hepatocyte like cells (HLCs) treated with Nitrofurantoin against control conditionWe wanted to investigate the transcriptional effect of Nitrofurantoin, a known drug induced liver injury (DILI)-compound, on hiPSC-derived HLCs. The goal was to identify which srress response pathways and biological processes are activated or altered in HLCs following Nitrofurantoin exposure. The compound is known to cause hepatotoxicity through mechanisms of oxidative stress, mitochondrial dysfunction, but the precise cellular responses in human liver models remain not fully understood.2025-08-09T00:00:00Z2025-07-25T11:44:34.639Z2025-07-25T11:44:34.639ZE-MTAB-15398ERP177266EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969