Psq hs96

Seven days after SNL or sham surgery, L5 DRG on the ipsilateral side was collected for DNA extraction. The treatment of genomic DNA with bisulfite was carried out using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA), according to the manufacturer's instructions. The region of the Kcna2 gene promotor and 5′UTR from the −540 to +500 bp site consisted of 65 CpG sites and were amplified. For the pyrosequence study, 4 pairs of primers of Kcna2 modified with 5′-Biotin (Supplementary Table 3) were used to amplify the bisulfite DNA. The master mix of the binding buffer, streptavidin-sepharose beads, and PCR products were then prepared for the binding reaction in a 96-well plate. The pyrosequence was performed using a PSQ HS96 (Biotage, Charlotte, NC) to determine percentage methylation at each CpG site. For the clone-sequencing study, the same primers without biotin modification were used in PCR amplification. The PCR products were purified and subcloned into pMD T-19 (Takara). After an overnight bacterial culture, 20 subclones from each PCR assay were subjected to direct sequencing.

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.

A unit of 100 ng of genomic DNA from each sample was treated with sodium bisulfate using an EZ DNA Methylation Gold Kit (ZYMO Research) following the manufacturer's protocol. The bisulfate-treated DNA was PCR amplified using unbiased nested primers. Quantitative pyrosequencing was performed using a PSQ HS96 (Biotage) to validate DMR regions. The DNA methylation percentage at each CpG site was measured using the Q-CpG methylation software (Biotage). SssI-treated human genomic DNA was used as 100% methylated controls and human genomic DNA amplified by Repli-G mini kit (Qiagen) was used as the non-methylated (0%) DNA control. Supplementary Table 15 provides the primer sequence used for the pyrosequencing reactions with the chromosomal coordinates in the University of California at Santa Cruz February 2009 human genome assembly (hg19) for each CpG site investigated.