Comparison of endogenous gene expression of HCT116 colorectal cancer cells and HCT116-derived DAPK1 knockout (ko) clones.

Data were obtained via Nanostring nCounter Gene Expression Assay (PanCancer Progression Panel, XT_PGX_HuV1_CancerProg_CSO XT-CSO-PROG1-12, Cat. no. 115000152, Nanostring Technologies, Hamburg, Germany) We aimed to reveal Death-associated protein kinase 1 (DAPK1)-dependent gene expression in the context of tumor progression covering genes involved in extracellular matrix, epithelial-mesenchymal transition, metastasis and angiogenesis comparing HCT116 DAPK1 wt cells and three different monoclonal DAPK1 ko clones named clone 7/6, clone 10/8 and clone 21/9. DAPK1 ko clones were generated by CRISPR/Cas9-mediated genomic editing. In this study, untreated biological replicates of HCT116 wt (n =3) and DAPK1 ko clones (clone 7/6: n = 2; clone 10/8: n = 3; clone 21/9: n = 1) were screened for gene expression of cancer progression related genes (770 genes including 30 housekeeping genes). Tumor cells were cultured in a 100 mm cell culture dish in RPMI 1640 medium supplemented with 1% penicillin/streptomycin and 10% FBS until 80% confluency and harvested via scraping off the plate. RNA was isolated from snap-frozen cell pellets. Gene expression analysis was performed using the human nCounter® PanCancer Progression Panel (NanoString Technologies). Total RNA was isolated from frozen cell pellets by QIAzol-chloroform extraction followed by RNeasy Mini Kit (Qiagen, Hilden, Germany) preparation. Isolated RNA (100 ng) was processed through the NanoString nCounter Prep Station. Briefly, the hybridization reaction (3 µl Reporter CodeSet, 5 µl hybridization buffer, 2 µl Capture ProbeSet and 5 µl total RNA) containing 100 ng RNA (HCT116: n = 3; clone 7/6: n = 2; clone 10/8: n = 3; clone 21/9: n = 1) was conducted for 16 h at 65 °C. Subsequently, samples were processed according to the manufacturer’s instructions and signals of reporter probes were counted and tabulated using the nCounter Digital Analyzer (NanoString Technologies). Finally, housekeeping gene (geometric mean of 30 genes) normalization for quantitating gene expression levels, positive control normalization for background noise correction and data analysis was performed using nSolver™ Analysis Software 3.0 (NanoString Technologies, Hamburg, Germany) and standard settings. The fold-change of counts was determined by averaging results per DAPK1 ko cell line and comparing them to average counts of HCT116 cells.