Identify the interplay between N6-methyladenine and gene regulation under hypoxia by randomized empirical model (ChIP-seq)

In this study, we employed ChIP-seq for histone modifications related to gene activation, such as H3K4me1, H3K4me3, and H3K27Ac, to investigate the role of N6-methyladenine (6mA) in chromatin accessibility under hypoxic conditions. Overall design: FaDu, a head and neck cancer cell line, was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1 mM sodium pyruvate, and 1× Antibiotic-Antimycotic (Gibco), under the conditions of 5% CO2 at 37°C. The mycoplasma absence was tested by Mycoplasma PCR Detection Kit (abm) before the experiments. Knockdown of METTL4 was achieved using a previously established method with lenti-virus infection. For hypoxic conditions, cells were incubated in an environment of 1% O2, 5% CO2, and 94% N2 at 37°C for 18 hours.

本研究采用针对与基因激活相关的组蛋白修饰（如H3K4me1、H3K4me3及H3K27Ac）的染色质免疫共沉淀测序（ChIP-seq）技术，探究N6-甲基腺嘌呤（6mA）在低氧条件下对染色质开放性的作用。
整体实验设计：
头颈癌细胞系FaDu购自中国台湾新竹生物资源保存及研究中心（BCRC）。将该细胞系培养于添加10%胎牛血清（FBS）、1 mM丙酮酸钠及1×抗生素-抗真菌混合液（Gibco）的达尔伯克改良伊格尔培养基（DMEM）中，于37℃、5% CO₂的培养条件下培养。实验开始前，采用abm公司的支原体PCR检测试剂盒完成支原体阴性检测。通过慢病毒感染法，采用已建立的实验方法实现METTL4基因的敲低。低氧处理组细胞置于含1% O₂、5% CO₂及94% N₂的培养环境中，于37℃孵育18小时。