Genomic DNA was extracted from seven cell lines using the Tissue DNA kit (Omega Bio-Tek, Inc.). Extracted DNA was bisulfite-modified using the BisulFlash DNA Modification kit (EpiGentek). The average methylation rates of 33 CpG sites that are located at −141551, −141494, −141439 and −141379 bp in the EPB41L3 promoter were detected. For EPB41L3, methylation rates of >20%, 10–20% and <10% were defined as hypermethylation, partial methylation, and non-methylation respectively (8 (link)).
For the methylation analysis, the extracted seven DNA samples were subjected to bisulfite conversion using the Bisulfite Conversion kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. Then, the purified bisulfate-converted DNA was used to perform MSP with biotinylated primers and Platinum Taq DNA polymerase (Kapa Biosystems, Inc., Wilmington, MA, USA). All the primers used for PCR were designed using PyroMark Assay Design 2.0 (Qiagen GmbH), synthesized by BGI (Shenzhen, China), and presented inTable II . The PCR products were purified using a Qiaquick PCR purification kit (Qiagen GmbH), and single-stranded DNA was prepared using Dynabeads M280 streptavidin (Thermo Fisher Scientific, Inc.). The samples were analyzed using PyroMark Q96 ID (Qiagen GmbH). Analysis of the results was performed with the PyroMark Q96-CpG software (Qiagen GmbH).
For the methylation analysis, the extracted seven DNA samples were subjected to bisulfite conversion using the Bisulfite Conversion kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. Then, the purified bisulfate-converted DNA was used to perform MSP with biotinylated primers and Platinum Taq DNA polymerase (Kapa Biosystems, Inc., Wilmington, MA, USA). All the primers used for PCR were designed using PyroMark Assay Design 2.0 (Qiagen GmbH), synthesized by BGI (Shenzhen, China), and presented in