Protective effect of UDCA against IL-11- induced cardiac fibrosis is mediated by TGR5 signalling
Protective effect of UDCA against IL-11- induced cardiac fibrosis is mediated by TGR5 signalling
Total RNA was isolated from cells with Trizol (Thermofisher Sci 15596018) on ice. Left ventricle (LV) free-wall tissue was minced into small chunks (1-2 mm3) and then digested in 0.05% Trypsin-EDTA (#25300054) for 2 min. Tissue chunks were then transferred into fibronectin-coated dishes (F1141) and cultured in DMEM supplemented with 20% FBS and 1% antibiotic-antimycotic at 37Â°C and 5% CO2. When confluent, fibroblasts were harvested from the dishes and used for experiments. Cultures were pre-treated for 24 hrs with UDCA before stimulation with 5ng/ml human IL-11 (PHC0115, Life Technologies) for a further 24 hrs.  Samples were purified with Monarch RNA Clean up kit (NEB, #T2030), including treatment with DNase I to eliminate any DNA contamination Purified RNA was given to the Imperial Genomics Facility who prepared the library and performed the sequencing experiments.  mRNA was selected by polyA selection and rRNA was depleted. Libraries were prepared using the TruSeq Stranded mRNACoda Sample Prep Kit (Illumina, California, USA).