All synthetic control DNA was obtained from Integrated DNA Technologies (IDT) and used at concentrations based on the manufacturer’s specifications. Human male genomic DNA (Promega) and EpiTect unmethylated control DNA (Qiagen) were used as unmethylated control genomic DNA. Enzymatically CpG-methylated HeLa genomic DNA (New England BioLabs) was used as a fully methylated control. Control genomic DNA was bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s protocol and eluted into volumes ranging from 12 to 25 μl. Post-bisulfite treatment yields were quantified by MethyLight using primer and probe sequences for β-actin recognizing both methylated and unmethylated templates: actin sense (5′-TAGGGAGTATATAGGTTGGGGAAGTT-3′), actin antisense (5′-AACACACAATAACAAACACAAATTCAC-3′), spanning a 103–base pair (bp) region (chr7:5,532,169-5,532,271), and 100 nM probe (5′-\56-FAM\TGTGGGGTG\ZEN\GTGATGGAGGAGGTTTAG\3IABkFQ\-3′. Assays were performed using 10× Master Mix to yield a final volume of 25 μl and final working concentrations of 16.6 mM (NH4)2SO4, 67 mM tris (pH 8.8), 6.7 mM MgCl2, 10 mM β-mercaptoethanol, 200 μM of each deoxynucleotide triphosphate (dNTP), and Platinum Taq polymerase (0.04 U/μl) (Thermo Fisher Scientific). Cycling conditions were 95°C for 5 min, followed by 50 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s. Standards for quantification were created by serial dilution of a 104-bp synthetic target equivalent to the bisulfite-converted locus.

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