2.6. RNA sequencing

Total RNA from cells treated with or without drugs for 24 h was isolated with RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The RNA integrity number (RIN) values were assessed with RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and were used for RNA quality evaluation. mRNA was extracted from the total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S, New England Biolabs, Ipswich, MA, USA). cDNA library was prepared from mRNA by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S, New England Biolabs). The library quality was evaluated using High Sensitivity DNA Analysis Kit on 2100 Bioanalyzer. RNA sequencing was performed with Illunima Hiseq 2500 (Illumina, San Diego, CA, USA). The raw data was analyzed with TopHat and Cuffdiff software15, 16. The gene expression profiles were uploaded to Reactome pathway database (https://reactome.org/) for signal pathway analysis.