2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
UOM-BCF
INSDC center name
public
INSDC status
ERS24435548
SRA accession
E-MTAB-15122:Sample 7
Submitter Id
ArrayExpress
broker name
EpH4
cell line
mammary epithelial cell
cell type
not collected
collection date
house mouse
common name
Protocols: 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.  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). 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). Library preparation of samples was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kits (Illumina).
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
3D
growth condition
soft
hydrogel stiffness
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
mammary gland
organism part
Mus musculus
scientific_name
Sample 7
title