Bisulphite pyrosequencing DNA methylation in the TRPA1 DMR genomic region was performed by a commercial laboratory service (EpigenDx). Briefly, 200–500 ng of genomic DNA was used for bisulphite modification using the Zymo Research EZ DNA Methylation Kit (Zymo Research), according to manufacturer’s instructions. Converted genomic DNA was PCR amplified using two sets of TRPA1 DMR unbiased primers, followed by pyrosequencing using PSQ HS96 (Biotage). Bisulphite pyrosequencing was performed as previously described59 (link). The Pyro Q-CpG methylation software (Qiagen) was used to determine the percentage of DNA methylation at each CpG site. Supplementary Table S8 provides the genomic location of the CpG sites measured in the TRPA1 CpG-island shore DMR. PCR reaction details and annealing temperatures for all bisulfite pyrosequencing reactions are also provided in Supplementary Table S8 .
Pyro q cpg methylation software
Manufactured by Qiagen
The Pyro Q-CpG methylation software is a tool developed by Qiagen for the analysis of DNA methylation patterns. It provides a platform for the quantitative assessment of DNA methylation levels at specific cytosine-phosphate-guanine (CpG) sites within a genomic region of interest.
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Lab products found in correlation
Psq hs96, by Biotage (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Pyromark gold 96 reagent kit, by Qiagen (1 mentions)
Annealing buffer, by Qiagen (1 mentions)
Vacuum prep tool, by Qiagen (1 mentions)
Streptavidin sepharose hp, by GE Healthcare (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Binding buffer, by Qiagen (1 mentions)
Pyromark id system, by Qiagen (1 mentions)
2 protocols using pyro q cpg methylation software
1
Bisulfite Pyrosequencing of TRPA1 DMR
2
Quantifying CpG site methylation via Pyrosequencing
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PCR products (20 μl) were added to a mix comprising Streptavidin Sepharose HP™ (3 μl; GE Healthcare, Dornstadt, Germany) and binding buffer (37 μl; Qiagen, Hilden, Germany). The contents were mixed at 2000g (Centrifuge 5810R, Eppendorf, Hamburg, Germany) for 10 min at room temperature. Using the Vacuum Prep Tool™ (Qiagen), according to the manufacturer's instructions, single-stranded PCR products were prepared. Sepharose beads with attached single-stranded templates were released into a PSQ 96 Plate Low™ (Qiagen) containing a mix of 40 μl annealing buffer (Qiagen) and the corresponding sequencing primer at 400 nmol l−1 (Supplementary Table 1
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