Pyromark assay design sw 2

DNA isolated from mouse lung tissues (100 ng) was treated with bisulfite using an EpiTect Bisulfite Kit (Qiagen, Frederick MD) according to the manufacturer’s instructions. DNA was amplified by PCR with primers for the following genes: Ahrr, DAPK1, CDH13, Tet1, and Rassf1. Bisulfite converted DNA was prepared for pyrosequencing according to the instructions in the PyroMark assay kit (Qiagen, Frederick, MD). Pyrosequencing was carried out according to the design files from Qiagen and the Qiagen PyroMark Assay Design SW 2.0 on the PyroMark Q96 (Qiagen, https://www.qiagen.com/us/products/discovery-and-translational-research/epigenetics/dna-methylation/pyrosequencing/software/pyromark-supplementary-software/). Primer sequences and experimental details are given in the Supplementary Methods.

The PCR primers 5′-gtaatttagtggtgttgttgaat-3′, 5′-biotin-cctaacccccaaccaacttcttactac-3′ and the sequencing primer 5′-gggttgagttaagtgtgtttggtaga-3′ for the region chr10:1405336-1405409 (hg19) were designed with Qiagen PyroMark AssayDesign SW 2.0. The samples were prepared with Qiagen EpiTect Fast Bisulfite Conversion and Qiagen PyroMark PCR kits and sequenced with Qiagen PyroMark Q24 Advanced system. The amplicons were analyzed with PyroMark Q24 Advanced software version 3.0.0. The statistical analysis was carried in R v3.4.3 [46 ] using packages lme4 v1.1-15 [20 (link)], car v2.1-6 [57 ], survival 2.42-4 [58 ], coxme 2.2-10 [59 ], and ggplot2 v2.2.1 [60 ]. Association of AD with CpG methylation was examined with lme4 package, with modeling methylation level as a function of disease status. Influence of covariates and their interaction with AD in the model was examined, including gender, age, zygosity, APOE genotype, and smoking (never, ever) as fixed effects and twin pair nested with genomic position and country of origin as random effects as specified in the results. The model with the lowest AIC was chosen. The Cox mixed effects model with coxme package was utilized to examine CpG methylation level as a prognostic marker of disease outcome. In this model, only age and gender were included as fixed effects and twin pair information nested with genomic position as a random effect.

Methylation quantification was carried out by pyrosequencing. Assays were designed using PyroMark Assay Design SW 2.0 (QIAGEN). Primers are provided in Table S4. Regions of interest were amplified from bisulfite converted DNA via PCR using biotinylated reverse primers and HotStarTaq DNA Polymerase (QIAGEN). The annealing temperature for PCR primers was optimized by gradient PCR. PCR conditions: 1) 95°C – 5 min; 2) 94°C – 30 s, optimized t°C – 30 s, 72°C – 55 s, 40 cycles; 3) 72°C – 5 min. PCR products were shaken at 1,400 rpm with Streptavidin Sepharose High Performance beads (GE healthcare) dissolved in binding buffer (10mM Tris-HCL pH7.6, 2M NaCl, 1mM EDTA, 0.1% Tween-20) for 20 min. The biotinylated strand was purified using the PyroMark Q96 Vacuum Workstation (QIAGEN). Sequencing primers were annealed to the template in annealing buffer (20mM Tris-acetate pH7.6, 2M magnesium acetate) at 85°C for 3 min. Sequencing was carried out on the PyroMark Q96 MD pyrosequencer (QIAGEN) using PyroMark Gold Q96 Reagents (QIAGEN).

Genomic DNA was isolated from cells using the QIAamp DNA Mini Kit (QIAGEN; cat. no. 51306) according to the user manual. The DNA was then subjected to bisulfite modification with the EZ DNA Methylation-Gold Kit (Zymo Research; cat. no. D5006) according to the instruction manual. The regions of interest were then amplified with primer pairs specifically designed to bisulfite-modified DNA, using the PyroMark PCR Kit (QIAGEN; cat. no. 978703) according to the user manual. Each pair of primers contained one biotinylated primer and one non-biotinylated one. The biotinylated ssDNA from each PCR amplicon was then isolated and subjected to pyrosequencing using the PyroMark Q24 System (QIAGEN; cat. no. 9001514) with a specific sequencing primer. The data were analyzed with Pryomark Q24 Software 2.0. All the PCR and sequencing primers were designed with the PyroMark Assay Design SW 2.0 (QIAGEN), and are listed in Supplement Table 5.

CpG methylation analysis of the replication cohort was performed by pyrosequencing. The assays were designed using PyroMark assay design SW 2.0 (Qiagen), and sequencing was performed using PyroMark Q24 (Qiagen) as previously described (34 (link)). The pyrosequencing primer sequences are listed in Supplementary Material, Table S3. For each sample, PCR reactions were formed in duplicate and the mean calculated, with samples being excluded from the analysis if the methylation between the PCR replicates differed by >5%. For each CpG site analysed, a Kruskal–Wallis test with Bonferroni correction for multiple testing was performed using GraphPad Prism software, and genotype–methylation correlations were deemed significant if the adjusted P-value was <0.05. Percentage methylation variability accounted for by genotype was calculated using a general univariate linear model with the SPSS Statistics software.

Primers for PCR and pyrosequencing were designed using PyroMark® Assay Design SW 2.0 (Qiagen). PCR was set up with 50 ng DNA template. PCR was a 25 μl MyTaq™ HS DNA Polymerase reaction (Bioline; London, United Kingdom) with 5x MyTaq Reaction Buffer (3 mM MgCl2, 1 mM dNTP), 0.4 µM forward and reverse primers, 5 U/µl MyTaq HS DNA Polymerase and dH₂O. The following PCR program was used: denaturation 95 °C 15 seconds, annealing 65 °C 30 seconds, elongation 72 °C 10 seconds for 30 cycles. The primer sequences have been provided in Supplementary Table S1. The products were analysed by standard 2.5% agarose gel electrophoresis. Assay set up was created using PyroMark® Q24 Software (Qiagen). Pyrosequencing was completed on PyroMark Q24 (Qiagen) according to the manufacturer instructions. The results were analysed using PyroMark® Q24 Software.

The gDNA (500 ng) was extracted from skeletal muscle specimens with the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. Bisulfite conversion was then performed using the EpiTect Fast DNA Bisulfite kit (QIAGEN). PyroMark Assay Design SW 2.0 (QIAGEN) was used to design the amplification and sequencing primers (Supplemental Table 7), and the methylation level of each CpG site was measured by pyrosequencing using PyroMark Q24 machine (QIAGEN).