Flexigene kit

SLC5A2 rs9934336 (c.127-121G>A, intronic single nucleotide polymorphism (SNP)) was selected based on previously published literature [14] (link). We also checked for other common putatively functional nonintronic SNPs in SLC5A2 using SNP database [18] (link) but no other candidate SNP was found.
Genomic DNA was extracted from whole-blood frozen samples collected at the inclusion in the study using the Qiagen FlexiGene Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Genotyping of SLC5A2 rs9934336 was carried out using a fluorescence-based competitive allelespecific (KASPar) assay according to the manufacturer's instructions (LGC Genomics, UK). Genotyping was performed blind to any clinical data and was randomly repeated in 15% of samples. Genotyping quality control criteria were 100% duplicate call rate and 95% SNP-wise call rate. Duplicate call rate and SNPwise call rate were 100%.

Genomic DNA was extracted from peripheral venous blood using FlexiGene Kit (Qiagen, Germany).

After thawing, the swim-up sperm fraction was purified by Silane-coated silica density gradients PureSperm 40/80 (Nidacon, Mölndal, Sweden). Purified sperm was resuspended in 300 μl buffer whose stock consisted of 5 ml of 5 M NaCl, 5 ml of 1 M Tris-HCl (pH 8), 5 ml of 10% SDS (pH 7.2), 1 ml of 0.5 M EDTA (pH 8), 1 ml of 100% β-mercaptoethanol and 33 ml of dH₂O. Upon addition of 100 μl proteinase K (20mg/ml), the samples were initially incubated for 2 h at 56°C (on a thermomixer). Additional 20 μl proteinase K was added and samples were incubated for another 2 h at 56°C. Subsequently, sperm DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the recommendations of the manufacturer. The FlexiGene kit (Qiagen, Hilden, Germany) was used for FCB DNA isolation. The concentration and quality of DNA were measured with the NanoDrop 2000c spectrophotometer (Thermo Scientific, Massachusetts, USA). Bisulphite conversion of sperm and FCB DNA (1 μg) was performed using the EpiTect Fast 96 Bisulphite kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol and the converted DNA was stored at -20°C. In our experience, the average methylation variation between the technical replicates (including bisulphite conversion, PCR, and pyrosequencing) is approximately 1–2 percentage points.

DNA was extracted from 300 μL frozen EDTA-whole blood using the Qiagen Flexigene kit (Qiagen, Venlo, Netherlands). Where frozen EDTA-whole blood samples were not available DNA was extracted from 200 μL frozen sera using the High Pure Viral Nucleic Acid kit (Roche Life Sciences, Basel, Switzerland) DNA was extracted according to the manufacturer’s instructions and eluted in 100 μL elution buffer and stored at minus 20 °C.