Pyromark q48 autoprep system

Bisulfite pyrosequencing was performed to confirm a peak in DNA methylation at the CpG site we identified in the D-LOOP (ChrM:545). Bisulfite conversion was performed using the Bisulfite-Gold kit (Zymo research, USA). A single amplicon (148bp) was generated using primers designed using the PyroMark Assay Design software 2.0 (Qiagen). DNA methylation was quantified using the Pyromark Q48 Autoprep system (Qiagen) following the manufacturer’s standard instructions and the PyroMark Q48 Autoprep 2.4.2 software. Bisulfite control regions showed a 97.5% conversion efficiency, with our 0% control showing methylation levels below the detection threshold.

Two CGi in 5′-flanking region (CGi1 SSC8:18527230–18528335; CGi2 SSC8:18524520–18526314) based on NCBI database and in exon 6 of PPARGC1A (CGi3 SSC8:17866933–17867385) based on ENSEMBL database, were selected and primers for CpG methylation analysis (Additional file 6) were designed using PyroMark Assay Design 2.0 software (Qiagen). DNA was purified from subcutaneous and visceral fat by phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich) extraction. Methylated and unmethylated controls were prepared with CpG Methyltransferase (Thermo Scientific) and REPLI-g Mini Kit (Qiagen), respectively. Five hundred nanograms DNA was bisulfite-converted using a EZ DNA Methylation-Gold Kit (Zymo Research). PCR reactions were performed on bisulfite-converted DNA using a Pyromark PCR Kit (Qiagen) according to manufactuer’s recommendations. Methylation analysis was carried out by pyrosequencing using Pyromark Q48 Advanced CpG reagents (Qiagen) and analyzed on Pyromark Q48 Autoprep system (Qiagen). CpG methylation level (%) was compared with Student t test between ASE samples (n = 5 for subcutaneous fat and n = 10 for visceral fat) and control samples (n = 10 for subcutaneous and n = 10 for visceral fat) with similar expression of both alleles.

Genomic DNA was extracted from subjects’ blood sampling. Methylation of 4 regions in SLC6A3 gene was assessed using bisulfite pyrosequencing. After bisulfite treatment of the DNA (EpiTect Fast DNA Bisulfite Kit, Qiagen, Hilden Germany), polymerase chain reaction test was done with DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad, CA, USA). Primer Design was designed with PyroMark Assay Design 2.0 (Qiagen, Hilden Germany). Pyrosequencing was done with PyroMark Q48 Autoprep System (Qiagen, Hilden Germany). PyroMark Q48 Autoprep 2.4.2 Software (Qiagen, Hilden Germany) was used to assess methylation of each CpG site, 4 regions in SLC6A3 gene. The percentage of methylated alleles was divided by the total number of alleles (methylated and unmethylated) to determine the level of methylation at each CpG site. The mean percentage of methylation (%) for each cluster was calculated by taking the average of all CpG site methylation levels measured within the cluster.

Genomic DNA was converted with bisulfite by using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. PyroMark software was used to design the pyrosequencing primers. In brief, bisulfite-converted DNA was subjected to PCR amplification in a total volume of 25 µL. DNA methylation was assessed using a PyroMark Q48 Autoprep system (QIAGEN) and PyroMark Q48 Advanced CpG Reagents (QIAGEN). The nucleotide dispensation order was generated by entering the sequences into the PyroMark Q48 Autoprep software ver. 2.4.2 (QIAGEN). The methylation at each CpG site was determined using the PyroMark Q48 Autoprep software set in CpG mode. The mean methylation of all CpG sites within the target region was determined by using the methylation at the individual CpG sites.

The UCSC genome browser was used to identify CGI regions in the MIR17HG and TET2 promoter regions (Figure 1). Pyromark Assay Design 2.0 software (Qiagen) was used to generate amplification and pyrosequencing primers to target regions in the CGI containing numerous CpGs (Table 4). Primers were designed to incorporate biotin on the reverse primer and an additional sequencing primer for the pyrosequencing, with one set targeting 7 CpGs in Exon 1 of TET2 and two regions of four CpGs were targeted within the promoter CGI of the MIR17HG gene, both regions that containing several transcription factor binding sites. Pyrosequencing was performed on a PyroMark Q48 Autoprep system (Qiagen) as per the manufacturer’s instructions using PyroMark Q96 Gold reagents (Qiagen). Pyrosequencing output analysis was performed using the Qseq software (BioMolecular Systems, V2.4.3).

Global DNA methylation was assessed by combining enzyme digestion with pyrosequencing using the LUminometric Methylation Assay (LUMA) as described previously^{57 (link)}, with minor modifications due to the PyroMark Q48 Autoprep System (Qiagen): the complete reaction digest was transferred to each well of a PyroMark Q48 Autoprep Disc, and 6.5 µl of PyroMark Annealing Buffer was added automatically by the instrument. The nucleotides were added to the cartridge chambers as follows: 50 μl dATPαS and 50 μl water to “A”, 50 μl dTTP and 50 μl water to “T”, 50 μl dCTP and 50 μl dGTP to “C”, and 100 μl water to “G”. Pyrosequencing was performed with the nucleotide dispensation order 5′-ACTCGA-3′.