Endogenous expression profiles of HCT116 colorectal cancer cells and HCT116-derived ATF2 knockout (KO) clones [Version 02]. Endogenous expression profiles of HCT116 colorectal cancer cells and HCT116-derived ATF2 knockout (KO) clones [Version 02]

Gene expression profiles were obtained via NanoString nCounter Gene Expression Assay (PanCancer Progression Panel, XT_PGX_HuV1_CancerProg_CSO, cat. no. XT-CSO-PROG1-12, NanoString Technologies, Hamburg, Germany). We aimed to decipher the role of activating transcription factor 2 (ATF2) in colorectal cancer, so-far masked by high intratumoral heterogeneity (ITH). To overcome ITH, we generated monoclonal ATF2 knockout (KO) cells (termed F9 and E5) using CRISPR/Cas9 gene editing and analyzed alterations of their gene expression profiles in comparison to the parental HCT116 cell line (HCT116 WT). Overall design: In this study, independent biological triplicates of HCT116 WT cells and ATF2 KO clones F9 and E5 (all n = 3) were investigated for gene expression alterations of genes related to tumor progression and metastasis using NanoString PanCancer Progression Panel (740 cancer-related genes plus 30 internal reference control genes). Tumor cells were cultured in a 10 cm cell culture dish in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin for 48 h until cells reached 80% confluency. Cells were harvested and total RNA was extracted from frozen cell pellets using a QIAzol-chloroform-based isolation and RNeasy Mini Kit (Qiagen, Hilden, Germany) for purification. Gene expression profiling was performed on 100 ng total RNA with the human nCounter PanCancer Progression Panel (NanoString Technologies). Briefly, hybridization reaction was performed on the NanoString nCounter® Prep Station for 17 h at 65 °C according to the manufacturer’s instructions. Samples were further processed according to the manufacturer’s protocol and signal of reporter probes were counted on the nCounter Digital Analyzer (NanoString Technologies). Gene expression data was further processed and analyzed using the package NanoStringDiff v. 1.14.0 (Wang et al., 2019) within the R v. 3.6.1 environment (R-Core-Team, 2019). Obtained fold changes (FC) were further screened for our selected cut-off criteria to finally extract an ATF2 loss-dependent gene signature. Endogenous expression profiles of HCT116 colorectal cancer cells and HCT116-derived ATF2 knockout (KO) clones [Version 02: all clones (n=6) vs WT (n=3)]