Illumina HiSeq 4000 paired end sequencing; Whole transcriptome analysis of rat peritoneal cells in a TGFβ1 induced mesothelial-to-mesenchymal transition
Whole transcriptome analysis of rat peritoneal cells in a TGFβ1 induced mesothelial-to-mesenchymal transition
Omental cells, from female Wistar rats weighing 220-250g (Charles River, Cambridgeshire, UK) were liberated by trypsin digestion (0.25% Trypsin-EDTA; Sigma-Aldrich, Dorset, UK) for 20min at 37°C. All subsequent procedures were carried out on ice.  Cells were incubated with anti-mesothelioma (HBME-1) primary IgM antibody (Dako, Cambridge, UK. 1:50 for 30min) in PBS, 3% BSA, similar to previously described [PubMed: 27403660]. Cells were washed with wash buffer (PBS, 10% foetal calf serum, FCS) and incubated with biotinylated secondary antibody (Vector Laboratories, Peterborough, UK. 1:100 for 30min) before further wash steps. Cells at a concentration of 1.5x106/80μl were incubated with Streptavidin-coated nanobeads (Biolegend, London, UK, 1:5) 15min. The cell suspension was then made up to a volume of 3ml with wash buffer and placed in the Mojo magnet (Biolegend) for 5min. Unconjugated cells were poured off and the sorted population re-suspended in cell culture media consisting of DMEM high glucose supplemented with 15% FCS, 4mM L-glutamine (Sigma-Aldrich), 1% v/v penicillin/streptomycin and 0.4μg/ml hydrocortisone (Sigma-Aldrich). Female Wistar rats weighing 220-250g (Charles River, Cambridgeshire, UK) were housed with free access to food and water under specific pathogen free conditions at the University of Manchester. Temperature 20-22 °C, Humidity 40-60%, Specified rodent pathogen free conditions, 12 hours light/dark cycle. Nutrients Rat and Mouse Standard Diet no.1 expanded (Ban Kingman).  Sorted mesothelial cells were seeded at 5x104/cm2 in multi-well plates. Adherent cells were apparent 48-72h post culture, at which point cells were washed with HBSS. To induce MMT, cells were placed in 5% serum containing media for 24h before exposure to 1ng/ml TGFβ1 (R&D systems, Abington, UK).  Cells were collected in RNA protect (Thermo Fisher Scientific, Runcorn, UK) and RNA extracted using the RNeasy Plus Mini Kit (Qiagen).  Truseq stranded mRNA Assay
Experimental Factor: stimulus
transforming growth factor beta 1