{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Leo Zeef"],"instrument_platform":["Illumina HiSeq 4000"],"study_type":["RNA-seq of coding RNA"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15122"],"description":["Almost all cells respond to the mechanical stiffness of their microenvironment through alter gene expression. Mammary epithelial cells respond to the mechanics of the extracellular matrix (ECM) in a way that can alter their behaviour and be pro-oncogenic. How increased ECM stiffness promotes transformation is unclear, but it can increase the incidence of DNA damage. This experiment was undertaken to identify changes in gene expression in non-tumorigenic mammary epithelial cells (murine Eph4) in response to different ECM stiffness. Epg4 cells were grown in either a soft or stiff 3D Matrigel-Alginate hydrogel, or on soft and stiff 2D polyacrylamide hydrogel. RNAseq was undertaken to identify gene expression changes associated with both stiffness and 2D vs. 3D culture conditions."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - EpH4 cells grown in hydrogels were extracted using excess 1X Trypsin-EDTA. Medium was removed from gels and then briefly washed with 1X PBS. Gels were then incubated for 5-10 minutes at 37°C with 2 ml 1X Trypsin-EDTA. The digested gel mixed was transferred to a 15 ml Falcon tube, and a further 2 ml 1X Trypsin-EDTA was added to the well and incubated for a further 5-10 minutes at 37°C. Digest mixes were pooled together and spun at 1000 RCF for 5 minutes. Pellets could then be resuspended for RNA extraction.","Nucleic Acid Extraction - RNA was isolated from EpH4 cells using Trizol. Briefly, cells were lysed directly with Trizol, and phase separated with chloroform. RNA was then precipitated with isopropanol and spun at 17000 rpm for 5 minutes and 4C. Pellets were resuspended in 5 M LiCl and kept at -20C for several hours before spinning for 20 minutes at 17000 rpm and 4C. Pellets were washed with 70 % ethanol, dried and resuspended in RNAse free water. Samples were assessed for concentration and purity by Nanodrop spectrometer 1000 (Thermo Scientific).","Sequencing - Samples were sequenced on an Illumina HiSeq 4000.","Library Construction - Library preparation of samples was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kits (Illumina).","Growth Protocol - There were 4 growth conditions; EpH4 cells were grown in either a 3D Matrigel-Alginate hydrogel (MAG) that was soft (0.3 kPa) or stiff (0.8 kPa) , or on 2D polyacrylamide hydrogel that was soft (0.4 kPa) or stiff (40 kPa). EpH4 murine mammary epithelial cells used in the experiments are described in https://doi.org/10.1007/s11626-998-0080-3. mRNA isolated from three independent replicates and gene expression across the transcriptome quantified. EpH4 cells were cultured in DMEM-F12 with 10% FBS (v/v), supplemented with 5 µg/mL insulin and 1% penicillin/streptomycin (v/v). For the experiment on hydrogels the cells were switched to a differentiation medium. Growth media was removed, and cells were washed briefly in PBS. Cells were then cultured on hydrogels for 10 days in differentiation medium (DMEM-F12, 10% FBS (v/v), 5 µg/mL insulin, 0.5 µg/mL hydrocortisone, 1% penicillin/streptomycin (v/v) and 3 µg/mL ovine prolactin)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Leo Zeef"],"additional_accession":[]},"is_claimable":false,"name":"RNAseq analysis of mouse Eph4 mammary epithelial cells to determine their response to altered extracellular matrix stiffness","description":"Almost all cells respond to the mechanical stiffness of their microenvironment through alter gene expression. Mammary epithelial cells respond to the mechanics of the extracellular matrix (ECM) in a way that can alter their behaviour and be pro-oncogenic. How increased ECM stiffness promotes transformation is unclear, but it can increase the incidence of DNA damage. This experiment was undertaken to identify changes in gene expression in non-tumorigenic mammary epithelial cells (murine Eph4) in response to different ECM stiffness. Epg4 cells were grown in either a soft or stiff 3D Matrigel-Alginate hydrogel, or on soft and stiff 2D polyacrylamide hydrogel. RNAseq was undertaken to identify gene expression changes associated with both stiffness and 2D vs. 3D culture conditions.","dates":{"release":"2025-10-22T00:00:00Z","modification":"2025-10-22T15:41:30.264Z","creation":"2025-05-06T21:34:50.162Z"},"accession":"E-MTAB-15122","cross_references":{"ENA":["ERP172312"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005518","EFO_0003738","EFO_0004184"]}}