RNAseq analysis of mouse Eph4 mammary epithelial cells to determine their response to altered extracellular matrix stiffness
RNAseq analysis of mouse Eph4 mammary epithelial cells to determine their response to altered extracellular matrix stiffness
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).