DNA methylation profiling (RRBS) of differentiating porcine synovial mesenchymal stem cells (PSMSCs) during the course of osteogenic induction
DNA methylation profiling (RRBS) of differentiating porcine synovial mesenchymal stem cells (PSMSCs) during the course of osteogenic induction
Porcine synovial mesenchymal stem cells (psMSCs) from the passage 2 were trypsinized, re-seeded at ~4 × 104 cells/ well or 2 × 104 cells / cm2 in a 24 well plate. The cells were induced to differentiate at 80-90 % confluence in osteogenic differentiation medium. The osteogenic differentiation medium contained 10% FBS, 5 µg/mL gentamycin, 100 nM dexamethasone (Sigma-Aldrich, St Louis, USA), 50 μM L-ascorbic acid (Sigma-Aldrich, St Louis, USA), and 10 mM β-glycerophosphate disodium salt hydrate (Sigma-Aldrich, St Louis, USA) in HG-DMEM. Cells were harvested for DNA isolation pre- (day 0) and post- osteogenic induction at 1, 7, 14 and 21 day. All cultures were maintained at 37 °C with 5% CO2 in a humidified atmosphere. Genomic DNA was extracted from samples using a QIAamp DNA MiniKit (Qiagen) with protenase K and Rnase A treatment according to the manufacturer's recommendation.  800 ng of DNA was spiked-in with 1% DNA control of unmethylated cl857 Sam7 Lambda DNA (Promega) and digested for 7 hours with 100U MspI (New England Biolabs) and then overnight with 50U TaqαI. The DNA fragments (double-enzyme digested) were end-repaired, A-tailed and ligated to TruSeq DNA adapters using a TruSeq Nano DNA library prep kit (Illumina) according to the manufacturer's protocols. Size-selection of the adapter-ligated DNA fragments were achieved by using sample purification beads (TruSeq Nano DNA library prep kit) to recover DNA inserts around 40–240bp. The adapter-ligated DNA libraries was subjected to a bisulfite conversion using an EZ DNA Methylation-Gold Kit according to the manufacturer's protocols (Zymo Research). Library amplification was performed using a PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of libraries was assessed on 2100 Bioanalyzer using a high-sensitive DNA chip (Agilent Technologies).