Methylated control dna

Genomic DNA was extracted from the ventricles of embryos at different developmental stages, and modified by the BisulFlash DNA Modification kit (34 (link)). CpGenome Universal Unmethylated DNA (sigma, S7822-M) and Methylated Control DNA (Sigma, M8570) were used as positive and negative controls, respectively. PCR primers for detecting the Hcn2-RE1 are listed in Supplementary Table S1. PCR fragments were gel purified using the MinElute Gel Extraction Kit and then cloned into the pCR2.1^® vector. After bacterial transformation, 6–10 positive colonies were randomly selected for bisulfite sequencing. The percentage of non-CpG methylation of each clone was calculated by the ratio of methylated sites to the total of non-CpG sites (for Hcn2-RE1, non CpG = 7). The average percentage of non-CpG methylation for the RE1 was then calculated from the triplicate assays.

DNA methylation analysis of specific sites was performed using
methylation-specific PCR (MSP). Bisulfite modification of genomic DNA (500ng)
was performed with the CpGenome™ Turbo Bisulfite Modification Kit (Merck
Millipore), according to the manufacturer's instructions. Each MSP reaction
incorporated 100ng of bisulfite-modified DNA, 1µL (10µM) of each
primer, and 1 × Go Taq Hot Start Green Master Mix (Promega Corporations,
Madison, WI, USA) in a final reaction of 25µL. Fragments were amplified
with specific primers for either methylated or unmethylated targets, as
previously described.^{24 (link),25 (link)} (Table 1). Methylated DNA (Methylated Control DNA, Sigma
Aldrich) and unmethylated DNA (CpGenome Universal Unmethylated DNA, Merck
Millipore) were modified, as previously described, and amplified by PCR as
control reactions with primers for the methylated and unmethylated conditions,
respectively. All reactions were performed in duplicate, and amplified PCR
samples (10µL) were loaded in 6% polyacrylamide gels and subjected to
electrophoresis. DNA bands were detected after silver staining.