NextSeq 550 sequencing; RNA-seq of control and CHH (cartilage-hair hypoplasia) fibroblasts transdifferentiated into chondrocytes-like cells
RNA-seq of control and CHH (cartilage-hair hypoplasia) fibroblasts transdifferentiated into chondrocytes-like cells
Dermal fibroblasts from control donors (n=4) and CHH patients (n=4) were isolated from skin biopsies at Freiburg University Hospital, Germany. Ethical approval for use of dermal fibroblasts was received from the medical ethical Institutional Review Board of Freiburg University Hospital. Informed consent was acquired from all subjects. Skin samples were cut into small pieces and cultured until fibroblasts were visible. Fibroblasts were then transferred into cell culture flasks. For fibroblast-derived chondrocytes (FDC) transdifferentiation, dermal fibroblasts were plated at high density into plates coated with aggrecan and cultured for three days in FDC transdifferentiation medium.  For fibroblast-derived chondrocytes (FDC) transdifferentiation, dermal fibroblasts were plated at high density  (200 000 cells/well) into 24-well plates coated with 5 μg aggrecan (Sigma-Aldrich) and cultured for three days in FDC transdifferentiation medium consisting of DMEM/F12 + Glutamax (Invitrogen), 10% FCS (Sigma-Aldrich), 1% antibiotic/antimycotic (Invitrogen), 1% NEAA (Thermo Fisher Scientific), 1% ITS (Insulin-Transferrin-Selenium-Sodium Pyruvate; Life Technologies, Carlsbad, California, United States), 50 μg/ml ascorbic acid 2-phosphate (Sigma-Aldrich) and 1 ng/ml human recombinant TGF-β3 (Life Technologies) as previously described. The medium was refreshed every other day. Total RNA was isolated at three different time points (Day 0, 1, and 3) using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific) according to manufacturers' protocol. RNA quality was assessed with an Agilents' 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent, Santa Clara, California, United States). Only samples with an RNA integrity (RIN) number greater than 9 were included for further analysis. TruSeq, according to manufacturers' protocol (Illumina).
Experimental Factor: disease
cartilage-hair hypoplasia
Experimental Factor: time
3