Bisulfite Conversion and Pyrosequencing of MSCs

DNA was isolated from Day0 MSCs and Day14 cartilagenous discs as above then 500 ng bisulphite converted with the EZ DNA Methylation Kit (Zymo Research, California, USA) following the manufacturer’s instructions. Pyrosequencing PCR was performed using the PyroMark PCR Kit (Qiagen) in a 20 µl reaction volume in duplicate with 1 µl bisulphite-converted DNA following the manufacturer instructions. Pyrosequencing assays were designed using PyroMark assay design software 2.0 (Qiagen), and the sequencing was performed using a PyroMark Q24 Advanced platform (Qiagen) with the recommended kit, following the instructions of the manufacturer and as previously detailed^{60 (link)}. Sequences of forward and biotinylated-reverse PCR primers and sequencing primers are listed in Supplementary Table 5.