The DNA extraction protocol was the same as the 850 K array screening subjects. DNA was bisulphite‐converted using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's standard protocol. Bisulphite‐converted DNA was amplified by polymerase chain reaction (PCR) using the PyroMark PCR Kit (Qiagen) in a total reaction volume of 25 μL, which contained sequencing primer (0.3 mmol/L) and 50 ng bisulphite‐converted DNA, with PCR primers listed in Table S1 . After purification, 20 μL PCR product was pyrosequenced using the PyroMark Gold Q96 Kit (Qiagen) and PyroMark Gold Q96 pyrosequencer (Qiagen) according to the manufacturer's instructions. Data were collected and analysed using the PyroMark Q96 software (version 2.5.8, Qiagen).
Pyromark gold q96 pyrosequencer
Manufactured by Qiagen
The PyroMark Gold Q96 pyrosequencer is a laboratory instrument used for DNA sequencing. It employs a pyrosequencing technique to determine the nucleotide sequence of a DNA sample. The instrument can process up to 96 samples simultaneously.
Automatically generated - may contain errors
2 protocols using pyromark gold q96 pyrosequencer
1
DNA Bisulfite Conversion and Pyrosequencing Protocol
2
Quantifying DNA Methylation via Pyrosequencing
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Two CpG sites were selected based on the results of the bisulfite sequencing analysis. Bisulfite-converted DNA was amplified by PCR with the reactions and conditions described in the PyroMark PCR kit. Pyrosequencing was performed using the PyroMark Gold Q96 pyrosequencer (Qiagen) according to the manufacturer’s instructions, and the percentage methylation per CpG was then calculated.
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