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

In this study, we employed RNA-seq 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RNA测序（RNA-seq）技术，探究缺氧条件下N6-甲基腺嘌呤（N6-methyladenine，6mA）对染色质可及性的调控作用。实验设计如下：人头颈部癌细胞系FaDu购自中国台湾新竹生物资源保藏与研究中心（Bioresource Collection and Research Center，BCRC）。该细胞系培养于添加10%胎牛血清（Fetal Bovine Serum，FBS）、1 mM丙酮酸钠以及1×抗生素-抗真菌混合液（Gibco）的达尔伯克改良伊格尔培养基（Dulbecco’s modified Eagle’s medium，DMEM）中，培养条件为37℃、5% CO₂。实验开展前，采用支原体PCR检测试剂盒（abm）对细胞进行支原体阴性检测。采用已报道的慢病毒感染法实现METTL4基因的敲低。缺氧处理组细胞置于37℃、含1% O₂、5% CO₂及94% N₂的培养环境中孵育18小时。