DNA was extracted from cells and tissues by classical phenol-chloroform method [5 (link)]. Bisulphite analysis of myogenin and PSEN1 promoter methylation was performed using the EpiTect Bisulphite kit; PCR products obtained after bisulphite treatment were cloned using the PCR Plus Cloning Kit (both from Qiagen). At least ten clones were analyzed per experimental condition using M13 primers for sequencing. Sequencing reactions of purified plasmid DNA were performed. Clones were sequenced by the cycle sequencing method using the ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Modified cytosine residues were recognized through comparison with the original DNA sequence and methylation status of any single cytosine in each sequenced clone were reported as 1/0 value in an excel spreadsheet (methylated: 1; unmethylated: 0). For each experimental sample we obtained the methylation % of each single cytosine by calculating the number of methylated cytosines divided by the number of clone sequenced per 100 ([n° methylC / n° sequenced clones] x100). Then, we calculated the average methylation % over the replicated cell culture experiments or over the 4 tissue samples for each experimental condition. Raw data related to DNA methylation results are shown in S5 Fig. GenBank accession numbers, primer names, sequence and position, expected products and annealing temperatures of the Methylation Insensitive Primers (MIPs) used for bisulphite analysis were already published [5 (link), 10 (link)]. Sequence, characteristics and position of both MIPs and MethPrimers are in S1 Fig. These primers allowed assessing methylation status of plus (5’->3’) DNA strand.
We also used different bisulphite modification assays as random control in samples characterized by low and high (CpG and non-CpG) methylation to ensure that cytosine conversion was complete. In particular, standard bisulphite procedures [8 (link)] with modifications previously described [10 (link), 29 (link)] and a modified method with ammonium bisulphite [30 (link)] were used. In all these cases the methylation patterns we found were similar. As negative controls of bisulphite modifications we used unmethylated purified PCR products of myogenin and PSEN1 promoter, obtained from genomic DNA as template with the same MIPs primers used for bisulphite PCR; the same purified PCR products where methylated in vitro with SssI methylase (New England Biochemistry), that methylates only cytosines in CpG dinucleotides, and were used as positive controls. We adopted all the possible cautions and controls to be sure that no methodological troubles could bias our analysis. In particular: 1) DNA samples to be compared were purified in parallel and modified in the same bisulfite assay; 2) amplifications with MIPs and with MethPrimers were performed on two aliquots of the same bisulfite-modified sample; 3) PCR products obtained by both MIPs and MethPrimers were always cloned in the same assay; 4) positive and negative controls were always used in each bisulfite assay; 5) clones were sequenced using two different instruments (the in-lab Applied Biosystems instrument and in service by Primm).
We also used different bisulphite modification assays as random control in samples characterized by low and high (CpG and non-CpG) methylation to ensure that cytosine conversion was complete. In particular, standard bisulphite procedures [8 (link)] with modifications previously described [10 (link), 29 (link)] and a modified method with ammonium bisulphite [30 (link)] were used. In all these cases the methylation patterns we found were similar. As negative controls of bisulphite modifications we used unmethylated purified PCR products of myogenin and PSEN1 promoter, obtained from genomic DNA as template with the same MIPs primers used for bisulphite PCR; the same purified PCR products where methylated in vitro with SssI methylase (New England Biochemistry), that methylates only cytosines in CpG dinucleotides, and were used as positive controls. We adopted all the possible cautions and controls to be sure that no methodological troubles could bias our analysis. In particular: 1) DNA samples to be compared were purified in parallel and modified in the same bisulfite assay; 2) amplifications with MIPs and with MethPrimers were performed on two aliquots of the same bisulfite-modified sample; 3) PCR products obtained by both MIPs and MethPrimers were always cloned in the same assay; 4) positive and negative controls were always used in each bisulfite assay; 5) clones were sequenced using two different instruments (the in-lab Applied Biosystems instrument and in service by Primm).
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