All the synthetic oligonucleotides used as primers were purchased (Integrated DNA Technologies, Tokyo, Japan). The coding-fragment of the RBM8A protein was amplified by PCR on cDNA prepared from MCF-7 cells using KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan) with the specific primer set (Table S3 ). The amplified DNA was subcloned into a TA cloning vector pGEM-T (Promega, Madison, WI, USA) using TaKaRa Ex Taq polymerase (Takara Bio, Kusatsu, Japan). The obtained pGEM-T-RBM8A plasmid was cleaved with Sfi I and subcloned into the Sfi I site of the pSBtet-GP vector (Addgene, Watertown, MA, USA) [25 (link)].
In order to establish stable cell lines, the pSBtet-GP constructs (pSB-DsRed and pSB-RBM8A) were co-transfected with the Sleeping Beauty transposon vector pCMV(CAT)T7-SB100X (Addgene) [26 (link)] using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. At 48 h post-transfection, cells were selected with 1 µg/mL puromycin (Sigma-Aldrich) for ~10 days and terminated when most cells showed GFP-derived green fluorescence. To induce RBM8A and the control DsRed proteins, 50 ng/mL of doxycycline (Takara Bio USA, Mountain View, CA, USA) was added to each cell line and cultured for 48 h.
In order to establish stable cell lines, the pSBtet-GP constructs (pSB-DsRed and pSB-RBM8A) were co-transfected with the Sleeping Beauty transposon vector pCMV(CAT)T7-SB100X (Addgene) [26 (link)] using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. At 48 h post-transfection, cells were selected with 1 µg/mL puromycin (Sigma-Aldrich) for ~10 days and terminated when most cells showed GFP-derived green fluorescence. To induce RBM8A and the control DsRed proteins, 50 ng/mL of doxycycline (Takara Bio USA, Mountain View, CA, USA) was added to each cell line and cultured for 48 h.