MCF-7 cells were treated with different compounds for 8 h to analyze differential gene expression. RNAseq of MCF-7 breast cancer cells

MCF-7 cells were grown in phenolred-free, high glucose DMEM medium, supplementent with 10% FBS and 1% Penicillin/Streptomycin. Cells were incubated at 37°C, 90% humidity, and 5% CO2. Cells were seeded 24 h before treatment. MCF-7 cells were treated for 8 h with the compounds of interest at a concentration of 25 µM. MCF-7 cells were further treated with 0.2% DMSO as control. Cells were harvested with phenolred-free trypsin and RNA extraction was performed with the InviTrap® Spin Cell RNA Mini Kit (Invitek Molecular GmbH, Berlin, Germany), according to the manufacturer’s instruction. RNA sequencing was performed by StarSEQ GmbH, Mainz, Germany. RNA quality was verified with a 2100 Bioanalyzer system. The NEBNext© Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, MA, USA) was used for mRNA preparation. Sequencing was carried out with the Illumina NextSeq 500™ system using 25 Mio paired-end reads (2 × 150 nt). Each sample was measured as biological duplicates.

MCF-7细胞于无酚红高糖DMEM培养基中培养，培养基补充有10%胎牛血清（FBS）及1%青霉素-链霉素混合液。细胞培养条件设定为37℃、90%相对湿度及5%CO₂。实验处理前24小时完成细胞铺板。MCF-7细胞采用浓度为25 μM的目标化合物处理8小时，同时设置0.2%二甲基亚砜（DMSO）对照组。使用无酚红胰酶消化收集细胞，并按照制造商说明书，采用InviTrap® Spin Cell RNA Mini Kit（德国柏林Invitek Molecular GmbH公司）进行RNA提取。RNA测序由德国美因茨的StarSEQ GmbH公司完成。采用2100生物分析仪系统验证RNA质量。使用NEBNext© Ultra™ II定向RNA文库制备试剂盒（美国马萨诸塞州纽英伦生物技术公司，New England Biolabs, MA, USA）完成mRNA文库构建。测序采用Illumina NextSeq 500™平台进行，获取2500万条双端测序读段（2×150 nt）。所有样本均设置两次生物学重复。