Global DNA Methylation and Hydroxymethylation Quantification

Genomic DNA from N. furzeri tissues was extracted using the Gentra Puregene Tissue Kit (Qiagen) according to manufacturer’s instructions and dot blots were done as previously described (Lagger et al., 2017). In brief, dot blots of genomic DNA and modified control oligonucleotides (unmethylated: oligo control, 5-methylated CpG: oligo 5mC, and 5-hydroxymethylated CpG: oligo 5hmC) were produced with Bio-Rad’s Bio-Dot Microfiltration apparatus according to manufacturer’s instructions. The control oligos (ordered from www.biomers.net) consisted of a synthetic sequence (49% GC content, 109bp in length) and T3 and M13-20 primer binding sites (underlined) with the following sequence: 5′-ATG​CTA​ATT​AAC​CCT​CAC​TAA​AGG​GAA​CTC​GAG​ACA​TCG​GAG​AAT​TCA​CAT​CAC​CGG​TGA​ATC​AGT​GCT​ACC​CGC​AAG​TGC​ACT​GGA​TCC​ACT​GGC​CGT​CGT​TTT​ACA​A-3′. Differentially methylated CpGs are highlighted in bold. Genomic DNA and oligonucleotide controls were denatured by boiling for 10 min at 100°C in 0.4 mM NaOH/10 mM EDTA in a total volume of 25 µl. Prior to spotting, DNA samples were neutralized by adding an equal volume of ice-cold 2 M ammonium acetate. Genomic DNA in technical duplicates (250 ng per well) and control oligonucleotides in a 1:2 dilution series (5 µM starting concentration) were spotted on a nitrocellulose membrane pre-soaked in 6x SSC buffer. After washing in 2x SSC, the membrane was crosslinked with a UV Stratalinker (Stratagene) and blocked in 5% non-fat dried milk powder in 0.1% Tween-20/1x TBS for 30 min at room temperature. The membranes were incubated with primary antibodies overnight at 4°C while rolling (5mC: 1:500 Eurogentec # BI-MECY-0100; 5hmC: 1:5.000 Active Motif # 39792). After incubation with secondary antibodies (anti-rabbit/anti-mouse IgG HRP conjugate, 1:10.000), 5mC and 5hmC signals were recorded using the ChemiDoc XRS + Imaging System (Bio-Rad) and analyzed with ImageLab Software (Bio-Rad). For normalization, the membranes were incubated with 0.02% methylene blue in 0.3 M sodium acetate pH5.2 for 5 min at room temperature, briefly destained in H₂O and imaged as described above. Levels of 5mC and 5hmC were normalized to the respective methylene blue signal intensities and calculated as mean of three biological replicates per time point. Raw values of the dot blot quantification are listed in Supplementary Table 4. Statistical significances were calculated with one-way ANOVA and multiple comparisons were performed by comparing the means of W11, W15, and W19 to W5 set to 1. ns: p > 0.05; *: p ≤ 0.05; **p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.