Sequenom massarray platform

To validate the results of pyrosequencing, the mtDNA methylation status of fourteen blood samples was reanalyzed using SEQUENOM MassARRAY platform (CapitalBio, Beijing, China). PCR primers were designed with Methprimer (http://epidesigner.com). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10-mer tag on the forward primer to adjust for melting temperature differences. Three pairs of primers were designed and used to amplify 3 fragments of mtDNA, from which 31 CpG sites were detected totally. The detailed information was listed in Supplementary Table 4. The bisulfite converted DNA was amplified using PCR Accesory set (Sequenom, San Diego, CA) and the amplification products were cleaned with shrimp alkaline phosphatase using Mass CLEAVE kit (Sequenom, San Diego, CA) to dephosphorylate all unincorporated nucleotides. After purification in vitro transcription of the reverse strand and base-specific (C or T) cleavage by RNase A was performed. The cleavage products, which differ in mass and length were spotted onto a MALDI matrix-containing SpectroCHIP and subjected to MALDI-TOF MS. The mass spectra was collected by MassARRAY Spectrometer and analyzed by EpiTYPER software version 1.0 (Sequenom, San Diego, CA).