{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Andrew Holding"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15331"],"description":["Estrogen receptor alpha (ERα) and hypoxia-inducible factors (HIFs) are key regulators of transcriptional programmes in breast cancer. To investigate their interplay, we performed RNA sequencing on ERα-positive breast cancer cell lines (MCF-7 and T-47D) following treatment with the selective estrogen receptor degrader fulvestrant and/or exposure to hypoxic conditions (1% O₂). Cells were cultured for 96 hours in the presence or absence of 100 nM fulvestrant, with the final 48 hours spent under normoxia or hypoxia. RNA was extracted from biological triplicates and sequenced using Illumina short-read technology (≥20 million reads/sample). Differential gene expression analysis revealed distinct transcriptional programmes regulated by ERα and hypoxia, as well as a large set of overlapping genes modulated by both conditions. Fulvestrant-induced ERα degradation suppressed canonical estrogen-responsive genes (e.g. GREB1, TFF1) and upregulated EPAS1 (HIF-2α). Hypoxia induced well-characterised HIF targets (e.g. CA9, VEGFA)."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Illumina-compatible RNA-seq libraries were prepared by the University of York Bioscience Technology Facility (BTF) Genomics Lab. Poly(A)+ RNA was isolated, fragmented, and reverse transcribed into cDNA. Libraries were constructed through end repair, A-tailing, adapter ligation, and PCR amplification following standard Illumina protocols.","Nucleic Acid Extraction - Total RNA was extracted using the Monarch Total RNA Mini Kit (New England Biolabs) according to the manufacturer’s instructions. RNA quality and concentration were assessed using an Agilent Bioanalyzer 2100 and Qubit fluorometer, respectively.","Sequencing - Libraries were sequenced on an Illumina NovaSeq 6000 platform (Azenta Life Sciences) using a 2 × 150 bp paired-end configuration. Base calling was performed using Illumina's real-time analysis software.","Sample Collection - MCF7 or T47D cells were seeded into 10 cm dishes and incubated at 37°C with 5% CO₂ for 24 hours. Cells were then treated with either 100 nM fulvestrant or ethanol vehicle control and maintained under normoxic conditions for 48 hours. Subsequently, dishes were either kept in normoxia or transferred to a hypoxic workstation for an additional 48 hours (1% Oxygen). At the end of the incubation period, cells were rinsed with PBS, trypsinised at 37°C for 5 minutes, and the trypsin was neutralised with culture medium. Cells were centrifuged at 300 × g for 5 minutes, washed once with PBS, and centrifuged again at 500 × g for 5 minutes. The resulting cell pellet was resuspended in PBS, treated with RNAlater (Sigma) for RNA preservation, and stored at −80°C until RNA extraction."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Raw read counts generated using the featureCounts() function from the Rsubread package were normalised and transformed using the DESeq2 package (v1.40.2) in R (v4.3.2). The DESeq2 pipeline was used to estimate size factors to account for library size differences across samples. Differential expression analysis was performed using the DESeq2 model.","Sequence Alignment - Raw paired-end RNA-seq reads (2 × 150 bp) were aligned to the human reference genome (hg38) using the Rsubread aligner (v2.14.2) in R. A genome index was first built using the buildindex() function with the hg38 FASTA file. Alignment was performed using align() with type set to \\\"rna\\\" and the reads sorted by genomic coordinates.  Only uniquely mapped reads were retained. The output was written in BAM format for downstream quantification."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Andrew Holding"],"additional_accession":[]},"is_claimable":false,"name":"RNA-Seq of ERα-positive breast cancer cell lines to measure the interplay of fulvestrant treatment and hypoxic conditions","description":"Estrogen receptor alpha (ERα) and hypoxia-inducible factors (HIFs) are key regulators of transcriptional programmes in breast cancer. To investigate their interplay, we performed RNA sequencing on ERα-positive breast cancer cell lines (MCF-7 and T-47D) following treatment with the selective estrogen receptor degrader fulvestrant and/or exposure to hypoxic conditions (1% O₂). Cells were cultured for 96 hours in the presence or absence of 100 nM fulvestrant, with the final 48 hours spent under normoxia or hypoxia. RNA was extracted from biological triplicates and sequenced using Illumina short-read technology (≥20 million reads/sample). Differential gene expression analysis revealed distinct transcriptional programmes regulated by ERα and hypoxia, as well as a large set of overlapping genes modulated by both conditions. Fulvestrant-induced ERα degradation suppressed canonical estrogen-responsive genes (e.g. GREB1, TFF1) and upregulated EPAS1 (HIF-2α). Hypoxia induced well-characterised HIF targets (e.g. CA9, VEGFA).","dates":{"release":"2025-07-21T00:00:00Z","modification":"2025-07-07T22:35:23.653Z","creation":"2025-07-07T22:35:23.653Z"},"accession":"E-MTAB-15331","cross_references":{"ENA":["ERP174928"],"EFO":["EFO_0002944","EFO_0004170","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}