Pyromark q48 autoprep software

Primers for bisulfite-specific PCR (BSPCR) were designed using the PyroMark Assay Design Software 2.0 (Qiagen) and with amplicon size ranging from 120– 250 base pairs (Supplementary Table 1. Bisulfite converted DNA was amplified by BSPCR using PyroMark PCR Mastermix (Qiagen). Briefly, 10ng of bisulfite converted DNA was mixed with the HotStarTaq Master Mix (Qiagen) and bisulfite specific primers under the following conditions: 95 °C for 10 min, 45 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s, and an elongation step of 72 °C for 10 min. The amplified products were run on 2% agarose gels to check the specificity of primers. Pyrosequencing was performed on Pyromark Q48 Autoprep using Q48 advanced CpG Reagents (Qiagen). Concisely, 10 μl of BSPCR product was added to 3 μl of magnetic beads and 2 μl of 4 μM sequencing primer on the Pyromark Q48 discs as per manufacturer’s instructions. Output data were analyzed using PyroMark Q48 Autoprep Software (Qiagen), which calculates the CpG methylation value as the percentage (methylated cytosine/ [methylated cytosine+ unmethylated cytosine]) for each CpG site, allowing quantitative comparisons. Controls to assess proper bisulfite conversion of the DNA were included in each assay to ensure the fidelity of the measurements. Analysis was performed on the average methylation of all CpGs of a particular gene taken together.

To selectively detect 5mC modification, genomic DNA was subjected to oxBS conversion (Booth et al., 2012 (link)) using TrueMethyl oxBS module (NuGEN Technologies) as per the manufacturer’s recommendations. In short, genomic DNA was affinity-purified using 80% acetonitrile (Fisher Scientific) and TrueMethyl magnetic beads to eliminate potential contaminating compounds. After the denaturation step, genomic DNA was oxidized to convert 5-hydroxymethylcytosine to 5-formylcytosine. Bisulfite treatment was then carried out to convert 5-formylcytosine to uracil, leaving 5-methylcytosine intact. Following desulfonation and purification, converted DNA was quantified using Qubit ssDNA assay (Invitrogen). PCR amplification of oxBS converted DNA was carried out with biotin-labeled primers. Primer design was carried out using PyroMark Assay Design software (version 2.0, QIAGEN). Pyrosequencing of biotinylated PCR products was performed using PyroMark Q48 Advanced CpG reagents (QIAGEN) on a PyroMark Q48 Autoprep apparatus (-QIAGEN) following the manufacturer’s protocol. 5mC levels at CpG sites were determined using PyroMark Q48 Autoprep software (version 2.4.2, QIAGEN) in CpG Assay mode. All samples were prepared, amplified and sequenced in triplicates. PCR and pyrosequencing primers are listed in Table S2.

For BSPCR, primers were designed using PyroMark Assay Design Software 2.0 (Qiagen), and bsDNA was amplified using HotStarTaq Master Mix (Qiagen) under the following conditions: 95°C for 10 min; 35 cycles of 94°C for 30 s, hybridization temperature for 30 s, and 72°C for 30 s; and elongation at 72°C for 10 min. The specificity of the primers was checked using horizontal agarose gel electrophoresis.
For quantitative analysis of each CpG in the candidate signature, the BSPCR products were used as templates for pyrosequencing on a PyroMark Q48 Autoprep system using PyroMark Q48 Advanced CpG Reagents (4 × 48; (Qiagen): 10 μl of the product was added to 3 μl of magnetic beads and 2 μl of sequencing primers (4 μM). Output data were analyzed using PyroMark Q48 Autoprep Software (Qiagen), which calculates CpG methylation values as (methylated cytosine/[methylated cytosine + unmethylated cytosine]), reflecting the methylation level on a scale from 0 (unmethylated) to 1 (fully methylated). To ensure the fidelity of the measurements, every assay contained a control for the bisulfite-mediated conversion of DNA.