mRNA expression profiling in MDA-MB-231 and HepG2 cells with 2 µM of ACP52C peptide treatment for 0, 12, 24, and 48 hours

The goal of this study is to compare mRNA expression profiles of the ACP52C-treated cells in time dependent manner. All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For the drug treatment, MDA-MB-231 and HepG2 cells were incubated in the presence of 2 μM ACP52C for 0, 12, 24, and 48 hours. Total cellular RNAs were extracted using Qiazol reagent. The RNA quality was examined by spectrophotometry, agarose gel electrophoresis (calculating the 18S and 28S rRNA ratio) and an Agilent Technologies 2100 Bioanalyzer (ensuring a RIN value greater than 7). The library was prepared using the RNA seq3’ mRNA-Seq Library Prep Kit FWD. The constructed libraries were subjected to 150-bp two-end sequencing using an Illumina NextSeq 500 sequencer at Insilicogen. All procedures were performed following the manufacturer’s instructions. Tophat 2 and cufflinks were used for reads mapping and expected read counts, respectively. DEGs were analyzed using an edgeR program and functional annotation of genes were achieved using a DAVID program. Duplicated mRNA profiles of ACP52C treated MDA-MB-231 and HepG2 cells are generated by 150 bp two end sequencing using Illumina NextSeq 500 and then analyze the differential expressed gene among these groups of cells.