Pyromark q24 vacuum workstation

Primers were designed for genes of interest using PyroMark Assay Design software (Qiagen, United States) (Supplementary Table S1). Bisulfite conversion of 40 ng of spermatic DNA was prepared by EZ DNA Methylation-Gold kit (Zymo Research, United States). Twenty ng of bisulfite converted DNA were amplified for pyrosequencing using Pyromark Q24 kit (Qiagen, United States) and Pyromark Q24 Vacuum workstation (Qiagen, United States) according to the manufacturer’s instructions.

Bisulfite conversion was performed using the EpiTect Plus DNA Bisulfite Kit (Qiagen) and 40 μL of the isolated cfDNA, according to the manufacturer’s instructions. Bisulfite-converted cfDNA was then eluted in 30 μL of elution buffer.
For the PCR amplification of the LINE-1 repetitive region using the PyroMark PCR Kit (Qiagen) 1 μL of bisulfite-treated DNA was used as the template. Samples along with methylated and unmethylated control DNA from the EpiTect PCR Control DNA kit (Qiagen) were run in triplicates. PCR protocol was as follows: initial denaturation at 95°C for 15 min; 50 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s; final extension was at 72°C for 10 min (Daskalos et al., 2009 (link)). The biotinylated PCR product was purified using the Pyromark Q24 Vacuum Workstation (Qiagen). Methylation levels of the six CpG’s were then measured by Pyromark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (Qiagen). cfDNA methylation levels were calculated as the ratio of C/T at a CpG site using the Pyromark Q24 Advanced Software 3.0.1 (Qiagen). Global cfDNA methylation was calculated as the average value of the six CpG’s. Primers used in cfDNA methylation analysis are listed in Table 4.

Bisulfite-converted DNAs were amplified by PCR primers designed with the Pyromark Assay Design software 2.0 (Qiagen, Hilden, Germany) and primers and cycling conditions for pyrosequencing shown in Supplementary table 2. These PCR amplicons were separated and detected by electrophoresis on 2% agarose gel. Briefly, the PCR amplicon for each sample was immobilized by master mix which contained Streptavidin Sepharose High Performance beads (GE, Healthcare) and PyroMark Binding Buffer (Qiagen). The immobilized product was purified by 70% ethanol, PyroMark Denaturation Solution (Qiagen), and PyroMark Wash Buffer (Qiagen) on the PyroMark Q24 Vacuum Workstation (Qiagen), sequentially. Finally, single-stranded DNA was then annealed to a specific sequencing primer (Supplementary table 2) at 80 °C for 2 min, and then cooled to room temperature for at least 5 min. Results were analyzed with the PyroMarkQ24 Software 2.0 (Qiagen).