Quantifying CpG Methylation in p16 and p21 Promoters

To quantify the CpG methylation status within p16^Ink4a and p21^Cip1/Waf1 promoters in MEFs, bisulfite conversion of genomic DNA was performed using an EZ DNA methylation kit (Zymo Research) as described previously [47 (link)]. PCR amplification of the targeted promoter regions was performed using the primers listed in Supplementary Table 1. After in vitro transcription using the PCR products as the template and T7 RNA polymerase, uracil-specific cleavage of the RNA transcripts by RNase A was conducted and the sizes of the resulting fragments were determined by MALDI-TOF mass spectrometry (MassARRAY Analyzer, Agena Bioscience, San Diego, CA, USA). The data were analyzed using an EpiTYPER software.