Analysis of DNA methylation in TET1 depleted colorectal cancer HCT116 cells

We aimed to analyze the relationship between TET1 and aberrant CpG methylation in colorectal cancer (CRC). We established two stable TET1 knockdown clones and negative control clones of HCT116 cells, and carried out DNA methylation analysis with HumanMethylation450 BeadChip. RNAi-induced TET1 knockdown was accomplished using a BLOCK-iT Pol II miR RNAi Expression Vector kit (Thermo Fisher Scientific). Two sets of oligonucleotides targeting TET1 were purchased from Invitrogen and ligated into a pcDNA6.2-GW/EmGFP-miR vector (Thermo Fisher Scientific). Cells were transfected with each TET1 knockdown vector or a pcDNA6.2-GW/EmGFP-miR-neg control plasmid (Thermo Fisher Scientific), after which they were selected with 0.6 mg/ml G418. GFP-positive colonies were isolated, and knockdown efficiencies were analyzed using RT-qPCR.