Analysis of DNA methylation in TET1 depleted colorectal cancer Colo320DM cells

We aimed to analyze the relationship between TET1 and aberrant CpG methylation in colorectal cancer (CRC). We established three stable TET1 knockdown clones and negative control clones of Colo320DM 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 1.0 mg/ml G418. GFP-positive colonies were isolated, and knockdown efficiencies were analyzed using RT-qPCR. Please note that the idat files linked as Series supplementary file contains raw data for multiple samples, as indicated in the corresponding sample description field.

本研究旨在分析结直肠癌（colorectal cancer, CRC）中TET1与异常CpG甲基化的关联。本研究构建了Colo320DM细胞的3株稳定TET1敲低细胞株及阴性对照细胞株，并采用人类甲基化450微珠芯片（HumanMethylation450 BeadChip）开展DNA甲基化分析。本研究采用BLOCK-iT Pol II miR RNAi表达载体试剂盒（Thermo Fisher Scientific）完成RNA干扰（RNA interference, RNAi）介导的TET1敲低实验：从Invitrogen公司购得2条靶向TET1的寡核苷酸链，并将其克隆至pcDNA6.2-GW/EmGFP-miR载体（Thermo Fisher Scientific）中。将细胞分别转染TET1敲低载体或pcDNA6.2-GW/EmGFP-miR-neg对照质粒（Thermo Fisher Scientific），随后使用1.0 mg/ml的G418进行抗性筛选。分离得到绿色荧光蛋白阳性细胞集落，并采用实时定量聚合酶链反应（real-time quantitative PCR, RT-qPCR）分析敲低效率。请注意：如对应样本描述字段所示，作为系列补充文件链接的idat文件包含了多个样本的原始数据。