Pyromark q24 advanced reagents

Genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, 69506) according to the manufacturer's instructions. A total of 200 ng of genomic DNA was bisulfite treated using the EpiTect Bisulfite kit (Qiagen, 59104) according to the manufacturer's instructions. This converts unmethylated cytosines to uracils. The bisulfite converted DNA was eluted with 20 μL elution buffer provided in the kit. Bisulfite PCR reactions for all genes described in this study were performed in a 25 μL volume containing 0.15 μL Hotstar Taq polymerase (5 U/μL) (New England Biolabs, M0495L), 2.5 μL 10× Standard buffer, 0.5 μL of 10 mM dNTPs, 1.0 μL of each primer (10 μM), and 1.5 μL bisulfite converted genomic DNA. PCR was performed under the following conditions: 95°C for 5 minutes followed by 45 cycles at 94°C for 30 seconds, 58°C for 1 minute, and 72°C for 45 seconds, and, finally, at 72°C for 7 minutes. The 4 μL PCR product was checked using gel electrophoresis. Pyrosequencing was performed with the PyroMark Q24 Advanced Reagents (Qiagen, 970922) using 20 μL PCR product from the bisulfite treated DNA and 20 μL sequencing primer (0.375 μM) according to the PyroMark Q24 CpG protocol. The general degree of cytosine methylation was determined by pyrosequencing of the bisulfite converted genomic DNA using the PyroMark Q24 Advanced System (Qiagen).

DNA bisulphite conversion was performed directly starting from cell pellets (a maximum of 1 × 10⁵ cells per sample) using the EZ DNA Methylation‐Direct kit (Zymo Research #D5021) following the manufacturer’s instructions. Target genomic regions were PCR amplified using 1 μl of converted DNA with biotin‐conjugated bisulphite primers (Table EV2), using the PyroMark PCR kit (Qiagen #978703). Pyrosequencing assay conditions were generated using the PyroMark Q24 Advanced 3.0 software and the sequencing reaction was performed with PyroMark Q24 advanced reagents (Qiagen, #970902) according to manufacturer’s instructions. Briefly, 10 μl of the PCR reaction was mixed with streptavidin beads (GE Healthcare #17‐5113‐01) by shaking for 5 min at room temperature and, after separation of DNA strands and release of samples into the Q24 plate (Qiagen) using PyroMark workstation (Qiagen), sequencing primers were annealed to DNA by heating at 80°C for 2 min and cooling down at RT for 5 min. Pyrosequencing was run on PyroMark Q24 advanced pyrosequencer (Qiagen) with target‐specific dispensation order (Table EV4). Results were analysed with PyroMark Q24 Advanced 3.0 software.