Unmethylated dna

The DNA extraction was performed from cryopreserved samples (-80 • C), using the kit Dneasy Tissue Kit QIAGEN ® (Qiagen, Chatsworth, CA), according to the manufacture's protocol. After the extraction, the DNA was treated with sodium bisulfite, which converts the unmethylated cytosines into uracil, as the methylated cytosines have been found to be resistant to this modification [13] . This reaction allows the identification of the gene methylation profile. 200 ng of DNA was amplified by Methylation-Specific PCR (MS-PCR). 0.4 M of each of the primers (Supplementary Table S2) were used to identify the methylated and unmethylated profiles, in distinct amplifications as described before and in [18] . Other products were employed in MS-PCR, including 0.2 mM of DNTP (Amersham Biosciences, Pittsburgh, PA, USA), 1X PCR buffer, 1 mM magnesium chloride, and 2.5 U of Platinum Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). Bisulfite-treated unmethylated DNA (Qiagen Inc., Valencia, CA, USA) was used as positive control of the unmethylated reaction. To obtain positive methylated controls, unmethylated DNA (Qiagen Inc., Valencia, CA, USA) was treated with MSssI methylase enzyme (New England Biolabs, Beverly, USA). The PCR products were analyzed by electrophoresis on 10% acrylamide gel stained with silver, and the methylation profile of each sample was observed.

Astandard curve was constructed using bisulfite-converted artificially methylated (Zymo Research) and unmethylated DNA (Qiagen) in different dilutions to acquire 0%, 0,1%, 0.5%, 1%, 5%, and 100% standards. The following primers were used for MS-HRM: SLC6A4 forward primer -CGAGGAGGCGAGGAGGTGT; SLC6A4 reverse primer -CGTTCCTCGTCTCCCACTCTAA (for chromosomal location and CpG sites see Suppl.
Figure S2). Methylation sensitive-high resolution melting experiments were carried out with the LightCycler 480 High Resolution Melting Master mix (Roche Diagnostics Schweiz AG, Switzerland), 250 nM primers and 1.5 µl of the bisulfite converted DNA, 2.5 mM Mg and water to complete the required volume. PCR and preliminary analysis of the data was conducted on the CFX96 real-time Thermal Cycler (Bio-Rad) and the Precision MeltAnalysis Software v1.2 (BioRad). For quantification of the data, a polyfit interpolating model in Matlab ® v.8.6 (Math Works) was used, according to a previously published protocol and algorithm obtained by the authors (Migheli et al., 2013) .