biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsBrian BwanyaN/AChromium™ Next GEM Single Cell 3′ Kit v3.1 (10X Genomics, PN-1000269)Illumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15516This study aimed to investigate how liver cell culture architecture influences acetaminophen (APAP)-induced transcriptional responses at single-cell resolution. We used human liver microtissues composed of primary human hepatocytes (PHHs), Kupffer cells (KCs), and hepatic sinusoidal endothelial cells (HSECs), cultured as 2D monolayers or 3D spheroids. Microtissues were exposed to vehicle, low-dose (350 µM), or high-dose (2687 µM) APAP for 24 hours. Single-cell suspensions were generated, and RNA libraries were prepared using the 10x Genomics Chromium platform, followed by Illumina sequencing. The resulting data were analyzed using Cell Ranger and Seurat to identify differentially expressed genes, assess pathway enrichment, and characterize cell-type–specific transcriptional responses to APAP exposure.biostudies-arrayexpressLibrary Construction - Single-cell RNA-Seq libraries were generated from cells using the Chromium™ Next GEM Single Cell 3′ Kit v3.1 (10X Genomics, PN-1000269) and the Dual Index Kit TT Set A, 96 reactions (10X Genomics, PN-1000215), following the manufacturer's protocol. Library concentration was measured with a Qubit 2.0 Fluorometer (ThermoFisher), and quality was assessed using an Agilent BioAnalyzer 2100.Nucleic Acid Extraction - After 24 hrs of exposure for 3D cultures, 192 spheroids per condition were collected in 900 microL PBS, washed and then resuspended in Liberase TL Research Grade™ 1.3 U/mL (Roche; 5401020001). For 2D, cells were treated with the same liberase digestion mix using 500 µL per well. The mixture was incubated at 37ºC for 30 min. shaking @100 RPM, with additional manual mixing by vortex every 10 min. Dissociation into single cells was checked using a microscope. Dissociated spheriods were kept on ice Cells were manually counted using Trypan blue and resuspended in PBS + 0.04 % BSA at a concentration of 1,000 cells/µL to recover ∼ 10,000 cells per sample.Sample Collection - Cryopreserved pooled plateable Primary Human Hepatocytes (PHHs), Hepatic Sinusoidal Endothelial Cells (HSECs) and male human Kupffer Cells (KCs) were obtained from Tebubio. All cryopreserved liver cells were thawed according to the suppliers’ instructions and resuspended in maintenance media (William’s E media with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml insulin, 5.5 μg/ ml transferrin, 6.7 ng/ml sodium selenite, 100 nM dexamethasone) supplemented with 20% foetal bovine serum (FBS). For 3D experiments, spheroids were established by seeding 1000 PHHs, 185 KCs and 250 HSECs per well in ultra-low attachment plates and centrifuged for 2 min at 125 × g. After 3 days media was changed to FBS-free maintenance media. For 2D experiments, 24-well plates (Gibco) were precoated with 5 µg/cm2 rat tail collagen type 1 in 0.02 M acetic acid for 1 hr at room temperature and washed 3 times with PBS. PHHs, KCs and HSECs in the same ratio as for 3D were seeded at 350,000 cells per well and allowed to attach for 4 hrs at 37 °C in a humidified incubator. Subsequently, debris was removed by shaking and washing the cells twice with maintenance media and cells were covered with 150 μL/well of 2 µg/mL collagen-mixture in DMEM and incubated at 37 °C for approximately 30 min until the collagen was fixed. Finally, media was replaced for maintenance media.Sequencing - Sequencing was carried out on an Illumina NovaSeq 6000 system using a 100-cycle S1 flow cell (v1.5) in paired-end mode.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesBrian BwanyaFlorian CaimentTwan van den BeuckenfalseSingle-Cell Transcriptomics of Acetaminophen-Induced Responses in Human 2D and 3D Liver MicrotissuesThis study aimed to investigate how liver cell culture architecture influences acetaminophen (APAP)-induced transcriptional responses at single-cell resolution. We used human liver microtissues composed of primary human hepatocytes (PHHs), Kupffer cells (KCs), and hepatic sinusoidal endothelial cells (HSECs), cultured as 2D monolayers or 3D spheroids. Microtissues were exposed to vehicle, low-dose (350 µM), or high-dose (2687 µM) APAP for 24 hours. Single-cell suspensions were generated, and RNA libraries were prepared using the 10x Genomics Chromium platform, followed by Illumina sequencing. The resulting data were analyzed using Cell Ranger and Seurat to identify differentially expressed genes, assess pathway enrichment, and characterize cell-type–specific transcriptional responses to APAP exposure.2025-09-01T00:00:00Z2025-09-01T01:03:05.754Z2025-08-29T05:26:40.777ZE-MTAB-15516ERP179376EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0004184