Pyromark q24 advanced cpg reagents

Bisulfite-treated DNA was amplified using PyroMark PCR kit (QIAGEN, Germany) (Table 2). PCR and sequence primers for pyrosequencing were designed with PyroMark Assay Design 2.0 (QIAGEN, Germany). Primer sequences are listed in Table 3. CAV1 methylation was analyzed by pyrosequencing using Pyromark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (QIAGEN, Germany) according to the manufacturer’s instructions. Briefly, in a skirted 24-well PCR plate 1 µl streptavidin beads (GE Healthcare, UK), 39 µl PyroMark binding buffer (QIAGEN, Germany), 20 µl high-purity water, and 20 µl PCR product were mixed and plate shook for a minimum of 10 min at 1400 rpm. By using the vacuum workstation, amplicons were denatured and transferred into a pyrosequencing plate containing sequencing primer. After heating the pyrosequencing plate for 3 min at 80 ^∘C, the plate was inserted into the pyrosequencer. Results were analyzed by the PyroMark Q24 Advanced Software 3.0.1. Analyzed sequence encompasses nine CpG sites (hg38; chr7:116,524,607-116,524,746) along EH38E2583972 ENCODE Candidate Cis-Regulatory Element (promoter). Average methylation of sample was calculated from nine CpG and also used in comparison. Which CpG from our study corresponds to which Illumina cg designations is listed in supplementary (Table S1).

Pyromark Q24 Advanced System using PyroMark Q24 CpG Advanced Reagents (#970922; Qiagen) was used for the pyrosequencing reaction, following the recommendation by Qiagen. 5′‐GGGGATTTAGTTTAGTGGT‐3′ was the sequencing primer for the ID element of the rat (Kim et al., 2007 (link)). DNA methylation data were obtained and analysed by the PyroMark Q24 Advanced Software. Quality control of the data was determined during the pyrosequencing run as described before (Mužić Radović et al., 2022 (link)).

PyroMark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (#970922; Qiagen) was used for the pyrosequencing reaction as recommended by Qiagen. 5’-GGGGATTTAGTTTAGTGGT-3’ was the sequencing primer for the rat ID element [21 (link)]. DNA methylation data were obtained and analyzed by the PyroMark Q24 Advanced Software.
Quality control of the data was determined during the pyrosequencing run. Quality control of bisulfite conversion for all samples was incorporated in the pyrosequencing assay. During assay design, the control dispensation of cytosine was generated at the non-CpG position. Since unmethylated cytosine should be converted to thymine, such control cytosine should not give a detectable signal [22 (link)]. The control of PCR reaction was conducted by running PCR blank, a reaction with all Master Mix components without the DNA. Samples obtained from PCR runs where blank control reactions did not show any product were considered for further pyrosequencing analysis. Moreover, the samples that passed quality control for bisulfite conversion after pyrosequencing were further analyzed.

Global methylation was measured in six samples from different animals per group by pyrosequencing. All steps were conducted as recommended by the manufacturer (Qiagen, Hilden, Germany). Pyromark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (#970922; Qiagen, Hilden, Germany) was used for the pyrosequencing reaction; 5′-GGGGATTTAGTTTAGTGGT-3′ was the sequencing primer for the rat ID element [46 (link)], while the data obtained were analyzed by the PyroMark Q24 Advanced Software. The global methylation was calculated as the average of the two analyzed CpG’s within the element [47 (link)].

Bisulfite conversion of 500 ng of DNA was done with EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol, after which the genomic regions of the BACH2, MGAT3, IL6ST, LAMB1 and HNF1A genes were amplified using a PyroMark PCR kit (Qiagen). Amplified genomic regions were then sequenced using the PyroMark Q24 Advanced pyrosequencing system (Qiagen) and PyroMark Q24 Advanced CpG Reagents (Qiagen) to quantify methylation level at individual CpG dinucleotides. Assay sequences are listed in Supplementary Table S1, and assay maps are shown in Figures 4, 5 and 6. Sequences of PCR and pyrosequencing primers are listed in Supplementary Table S2.

PSQ was performed using the PyroMark Q24 Vacuum Workstation and PyroMark Q24 Advanced instrument with PyroMark Q24 Advanced CpG Reagents and PyroMark Q24 Advanced Accessories (all Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Primer sequences, sequences to analyze, and dispensation orders are listed in Table 1. DNA immobilization was optimized in a range from 5–39 µL biotinylated PCR-HRM product (pool of technical replicates) with 1 µL Streptavidin Sepharose High Perfomance beads (GE Healthcare, Germany) and 40 μL PyroMark Binding Buffer per 80 µL immobilization reaction. 22.5 µL biotinylated PCR-HRM product, 1.5 µL Streptavidin Sepharose High Performance beads, and 60 μL PyroMark Binding Buffer per 120 µL immobilization reaction was also tested. DNA immobilization was performed under agitation for 10 min at 1400 rpm. The captured PCR product was denatured, washed, and finally the biotinylated strand was transferred into a PyroMark Q24 Plate containing 20 µL of 0.375 μM sequencing primer in PyroMark Annealing Buffer. The plate was heated at 80 °C for 5 min and then transferred into the instrument holding the PyroMark Q24 Cartridge loaded according to pre-run information provided by the PyroMark Q24 Advanced software 3.0.0 (Qiagen, Hilden, Germany).

Primers for pyrosequencing methylation assays of DMRs were designed using the PyroMark Assay Design 2.0 Software (Qiagen, NL). For repetitive regions, we could not anchor primers to non-repetitive flanking sequences because the closest non-repetitive regions were farther than two kb from the differentially methylated CG sites. Hence, both PCR primers were located within the repeat and, in principle, could amplify more than one copy of the transposon. The list of primers for pyrosequencing methylation assays is provided in Table S3.
250 to 1000 ng of DNA per sample were treated with sodium bisulfite using EpiTect Bisulfite Kit (Qiagen, NL). Pyrosequencing was carried out using the PyroMark Q24 Advanced platform and PyroMark Q24 Advanced CpG Reagents (Qiagen, NL). Results were analyzed using the PyroMark Q24 Advanced software (Qiagen, NL).