Pyromark q48

DNA was extracted from 25 mg of snap frozen liver using the QIAmp mini kit (QIAGEN, Hilden, Germany). Genomic DNA was bisulphite-converted (bisDNA) using the EpiTectFast Bisulfite kit (QIAGEN). Bisulphite PCR was performed with the PyroMark PCR kit (QIAGEN). One primer pair was used to analyse CpG sites 1–5, a second primer pair was used to analyse CpG site 6 and a third pair was used for analysis of cg25924746 (see ESM Table 2 for a list of primers, including primer for rs4547213). DNA methylation was measured by bisulphite pyrosequencing using PyroMark Q48 and PyroMark Q48 Advanced reagents (QIAGEN) (see ESM Methods).

We used bisulfite pyrosequencing to quantify the degree of DNA methylation at eight CpG sites upstream and six CpG sites downstream of the CTG repeat in the 3′ UTR region of the DMPK gene (Figure 1). These regions were chosen as previous studies had associated the methylation of these sites with the age at disease onset [4 (link),19 (link)] and the clinical phenotype [20 (link)]. Bisulfite conversion was carried out on 200 ng of DNA with an EZ-DNA Methylation-Gold kit (Zymo Research, Irvin, CA, USA), according to the manufacturer’s instructions, with an elution volume of 10 μL. Methylated and non-methylated standards and a no template control (NTC) were included with every conversion. PCR was performed with a Pyromark PCR kit (Qiagen, Hilden, Germany), using 1 μL of bisulfite-converted DNA. The PCR primers were designed with PyroMark Assay Design 2.0 (Qiagen) and were targeted to the regions flanking the CTG repeat (Figure 1). The PCR products were visualized on an agarose gel, and the methylation levels were quantified using a Pyromark Q48 Autoprep (Qiagen) and the Pyromark Q48 software (Qiagen). The primer sequences, modifications to the manufacturer’s instructions, and PCR conditions can be found in Table S1.