Pyromark assay design 2

DNA from the same samples as above were subjected to bisulfite conversion using the Zymo EZ DNA Methylation Kit (Zymo Research, Irvine, California), which converts DNA methylation information into sequence base differences by deaminating unmethylated cytosines to uracil while leaving methylated cytosines unchanged. Bisulfite pyrosequencing assays were designed with PyroMark Assay Design 2.0 (Qiagen, Hilden, Germany; Supplementary Table 4). The regions of interest were amplified by PCR using the HotstarTaq DNA polymerase kit (Qiagen, Hilden, Germany) as follows: 15 min at 95°C, 45 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and a 5 min 72°C final extension step. For pyrosequencing, single-stranded DNA was prepared from the PCR product with the Pyromark™ Vacuum Prep Workstation (Qiagen, Hilden, Germany) and the sequencing was performed using sequencing primers on a Pyromark™ Q96 MD pyrosequencer (Qiagen, Hilden, Germany). The quantitative levels of methylation for each CpG dinucleotide were calculated with Pyro Q-CpG software (Qiagen, Hilden, Germany). Of note, only PAE and C animals were assessed by bisulfite pyrosequencing. We selected several DMRs for verification by bisulfite pyrosequencing based on their potential role in PAE-induced deficits, mainly focusing on their associated gene.

DNA was extracted from DRG of diabetic and control rats and underwent bisulfite conversion per manufacturer’s instructions (Cat#D5020, Zymo Research, USA). Biotinylated primers and the pyrosequencing assays were designed using PyroMark Assay Design 2.0 (Qiagen, Inc.) software to cover 7 CpG sites on IGF-1 promoter. PCR and pyrosequencing performed as previously described [35 (link)]. Sequencing primers were then added for pyrosequencing per manufacturer’s instructions (Pyromark™ Q96 MD Pyrosequencer, Qiagen, Inc.). See Supplementary Information for more details.

Ten-micron sections were utilized to make DNA from each block. DNA isolation and sodium bisulfite modification were performed according to the manufacturer’s protocol using the EpiTect Plus FFPE Bisulfite Kit (Qiagen, CA, USA). Bisulfite-modified DNA was then amplified using PCR in preparation for pyrosequencing, with either biotinylated forward or reverse primer. All PCR and sequence primers for pyrosequencing were designed using PyroMark Assay Design 2.0 (Qiagen), have been previously described (4). PCR products were captured with streptavidin sepharose beads, denatured to single strand, and annealed to the sequencing primer for the pyrosequencing assay. Human Premixed Calibration Standard with different percentage of methylation (EpigenDx, Hopkinton, MA), human white blood cell DNA, and SssI methylase-treated DNA from human PC cells-Du145 were used as controls in each run. Methylation was quantified with the PyroMark MD Pyrosequencing System (Qiagen) within the linear range of the assay. All samples were analyzed using two independent experiments.

DNA was isolated from untreated- and 5-aza-dC; (BON-1: EC₅₀ 100 nM, QGP-1: EC₅₀ 50 nM) treated BON-1 and QGP-1 cells, according to protocol with the Genome Wizard DNA isolation kit (Promega Corporation, Madison, USA). For bisulfite conversion 1000 ng input DNA was used with the Zymo Research EZ DNA Zymo kit according to manufacturer’s protocol (Zymo Research Corporation, Irvine, USA). Primer design was done with PyroMark Assay Design 2.0 (Qiagen N.V., Venlo, the Netherlands). Bisulfite treated DNA was aliquoted and stored at -20°C. PCR of bisulfite treated DNA was performed with the primers listed in Table 1 and with the following program: 5 minutes 95°C, 45 cycles 30 seconds 95°C - 30 seconds 60°C – 30 seconds 72°C, 1 cycle 10 minutes 72°C, ∞ 4°C. PCR products were analyzed on the PyroMark Q24 (Qiagen) with PyroMark Gold Q24 reagents (Qiagen) according to manufacturer’s protocol

Quantitative DNA methylation analysis of the TMPRSS4 promoter was performed by bisulfite pyrosequencing of two consecutive potentially methylated cytosines located at −116 bp and −99 bp relative to the TSS. Bisulfite conversion of 500 ng of each DNA sample was performed with EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer's recommendations. Primer sequences (Supplementary Table 4) were designed with PyroMark Assay Design 2.0 (Qiagen). None of the PCR primers covered any CpG. PCR for TMPRSS4 promoter was performed with 1 μl of bisulfite converted DNA with biotinylated primers using an annealing temperature of 60°C and 50 cycles. PCR products were verified on 2% agarose gels before pyrosequencing analysis. Pyrosequencing was performed using a Pyro Gold SQA™ Reagent Kit (Qiagen) in a PyroMark Q96 System version 2.0.6 (Qiagen) according to the manufacturer's instructions. CpG site methylation quantification was obtained using Pyro Q-CpG 1.0.9 (Qiagen).

Bisulphite pyro-sequencing was performed as described previously [11 (link)]. Briefly, 0.5 μg of genomic DNA was bisulfite-modified using the EZ DNA Methylation Kit (Zymo Research, Orange, CA), according to the manufacturer’s protocol. The bisulfite-modified DNA was subjected to PCR amplification using a tailed reverse primer in combination with a biotin-labeled universal primer. The PCR and sequencing primers were designed using PyroMark Assay Design 2.0 (Qiagen GmbH, Hilden, Germany). The ABCA1 TSS (−90 to +190) was PCR-amplified with specific primers (Additional file 1: Table S1) in a 25-μl reaction containing 2× RBC SensiZyme Hotstart Taq premix (RBC Bioscience, Taiwan). Prior to pyro-sequencing, 1.5 μl of each PCR reaction was analyzed on 1% agarose gel. The pyro-sequencing was performed on the PyroMark Q24 instrument (Qiagen) using the Pyro Gold Reagents (Qiagen), according to the manufacturer’s protocol. The methylation level of 11 CpG sites, which are located −13 to +95 with respect to the TSS, was measured. The methylation percentage of each cytosine was determined using the fluorescence intensity of cytosines divided by the sum of the fluorescence intensity of cytosines and thymines at each CpG site. In vitro methylated DNA (Millipore) was included as positive control for pyro-sequencing.

Four different pyro-sequencing assays were designed to properly determine the genotype at any of the four SNP positions associated with the Sex-linked barring phenotype. The primers were designed using PyroMark Assay Design 2.0 (Qiagen). cDNA or DNA samples were amplified as outlined in the Supplementary section. The obtained PCR products were purified and transferred to a PyroMark Q96 MD pyro-sequencing machine (Qiagen). In short, 20 μl of PCR product were hybridized to Streptavidin Sepharose High performance beads (GE Healthcare) and washed using 70% ethanol (Solveco), denatured in 0.2 M NaOH and washed again in wash buffer (composition as described in the pyro-sequencing manual) followed by a hybridization for 2 min incubation at 82°C.