Gipz lentiviral shrna

GIPZ lentiviral shRNA (cat #V3LHS_641817 and #V3LHS_641819 clones purchased from Thermo Scientific) was used to establish STAT3 knockdown in HTOXAR3 colorectal cell lines. Glycerol-stored plasmid was previously replicated, isolated, transfected in HEK293T cells and finally transduced in target cells. Cells were selected, according to kill curve values, with puromycin (2 μg/ml). A non-silencing GIPZ lentiviral shRNA containing a random vector was used as a control in both cell lines in subsequent experiments. Western blot was performed to confirm STAT3 silencing efficiency in colorectal cell lines.

Lentivirus-mediated shRNA against the mouse Dnmt1 gene (GIPZ lentiviral shRNA) and a shRNA control (GIPZ lentiviral empty vector shRNA) were purchased from Thermo Fisher Scientific. Preparation of organoids and lentiviral infection were performed as described previously14 (link). Twenty-four hours after transduction, the infected organoids were selected with puromycin (1 μg/ml).