biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsMorgan HowellsNextSeq 500RNA-seq of coding RNA from single cellsHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16057This experiment investigates the transcriptional landscape of LNCaP prostate cancer cells in response to hormonal and epigenetic therapy under hypoxic conditions using single-cell RNA sequencing (scRNA-seq). The study aims to model drug resistance mechanisms by exposing cells to chronic hypoxia and treating them with Enzalutamide, an androgen receptor inhibitor, with and without Tazemetostat, an EZH2 inhibitor. Cells were sampled across multiple time points to identify resistant clones and assess how combination therapy alters gene expression. This experiment provides insight into the gene expression signatures associated with resistance and explores potential biomarkers relevant to advanced prostate cancer progression.biostudies-arrayexpressLibrary Construction - cDNA synthesis and library preparation were performed using the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 (PN-1000268/1000269) together with the 3′ Feature Barcode Kit (PN-1000262), Chromium Next GEM Chip G Single Cell Kit (PN-1000127/1000120), and Dual Index Kit TT Set A (PN-1000215). Libraries were constructed according to the manufacturer’s instructions at the Babraham Institute Genomics Facility.Sample Collection - LNCaP human prostate cancer cells were cultured under chronic hypoxia (2% O₂) in RPMI-1640 medium (Gibco #21875034) supplemented with 10% fetal bovine serum (ThermoFisher #26140079) and Penicillin/Streptomycin/Amphotericin B (ThermoFisher #15140122). Cells were harvested by trypsinization at experimental time points (Day 3, Day 6, Day 9), counted using a Luna-FL cell counter, and assessed for viability via trypan blue exclusion. Samples were maintained at 2% O₂ until processing.Nucleic Acid Extraction - Single-cell suspensions were prepared following the 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1 user guide. RNA molecules were captured within Gel Beads-in-Emulsion (GEMs) using barcoded oligo-dT primers. Reverse transcription within droplets generated cDNA tagged with unique molecular identifiers (UMIs) and cell barcodes. No separate bulk RNA extraction step was performed.Sequencing - Libraries were quality-checked using an Agilent 2100 Bioanalyser (High Sensitivity DNA Assay) and quantified using the KAPA Biosystems Library Quantification Kit (Roche). Pooled libraries were sequenced on an Illumina NextSeq 500 using a High Output kit, producing approximately 500 million reads with a Q30 ≥ 92%. Sequencing was carried out as single-end reads according to the 10x Genomics 3′ RNA-seq configuration.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMorgan HowellsfalseSingle-cell RNA-seq of LNCaP prostate cancer cells treated with Enzalutamide and Tazemetostat under hypoxic conditionsThis experiment investigates the transcriptional landscape of LNCaP prostate cancer cells in response to hormonal and epigenetic therapy under hypoxic conditions using single-cell RNA sequencing (scRNA-seq). The study aims to model drug resistance mechanisms by exposing cells to chronic hypoxia and treating them with Enzalutamide, an androgen receptor inhibitor, with and without Tazemetostat, an EZH2 inhibitor. Cells were sampled across multiple time points to identify resistant clones and assess how combination therapy alters gene expression. This experiment provides insight into the gene expression signatures associated with resistance and explores potential biomarkers relevant to advanced prostate cancer progression.2025-08-07T00:00:00Z2025-11-13T17:43:03.788Z2025-11-13T13:19:04.765ZE-MTAB-16057ERP184021EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0004184