Plasma DNAs were isolated using QIAmp UltraSense Virus Kits (Qiagen, Valencia, CA). Aliquots of DNA (500 ng) were bisulfite-treated with the EZ DNA methylation kit (Zymo Research, Orange, CA) following the manufacturer’s protocol. Pyrosequencing for LINE-1 methylation levels was carried out using PCR and sequencing primers as described previously [5 (link)]. Briefly, PCR was carried out in a 25 μl reaction mix containing 50 ng bisulfite-converted DNA, 1X Pyromark PCR Master Mix (Qiagen), 1X Coral Load Concentrate (Qiagen) and 0.2 μM forward and reverse primers. Following amplification, the biotinylated PCR products were purified and incubated with the sequencing primer designed to bind adjacent to the CpG sites of interest. Pyrosequencing was conducted using a PyroMark Q96 instrument (Qiagen), with subsequent quantitation of methylation levels determined with the PyroMark CpG 1.010 software. Relative peak height differences were used to calculate the percentage of methylated cytosines at each given site. Percent methylation within a sample was subsequently determined by averaging across all three interrogated CpG sites in the analysis.