Turbofectin transfection reagent

The DDR2 expressing plasmid and its empty plasmid pCMV6-XL6 were both obtained from Origene Technologies Inc. (Rockville, USA). DDR2 expressing plasmid or empty plasmid pCMV6-XL6 was transfected into Hep3B cells using TurboFectin Transfection Reagent purchased from Origene Technologies Inc. (Rockville, USA). The DDR2 shRNA and scrambled shRNA vector pRS were also purchased from Origene Technologies Inc. (Rockville, USA). DDR2 unique 29mer shRNA constructs in pRS Vector or scrambled shRNA vector pRS was transfected into MHCC-97H cells using TurboFectin Transfection Reagent purchased from Origene Technologies Inc. (Rockville, USA). The specific siRNA sequences against ERK2 (sc-35335), against SNAIL1 (sc-38398) and the scramble siRNAs (sc-37007) were obtained from Santa Cruz Biotechnology (Santa Cruz, USA).

The METTL7A Human shRNA Plasmid Kit (Origene, Cat. TF311498, MD, USA) and pRFP-C-RS Vector (Origene, Cat. TR30014, MD, USA) was used. The plasmid contains the sequence that inhibits METTL7A mRNA and the restriction enzyme sites that are responsive to chloramphenicol and puromycin. To confirm the transfection efficiency, the RFP gene was inserted into the vector. The pRFP-C-RS shRNA vector was used as the control. Transfection was performed using 2.5 µg of TurboFectin transfection reagent (Origene, Cat. TF81001, MD, USA) mixed with 2 × 10⁵ cells according to the manufacturer’s instructions. Transfected cells were cultured for 2 days at 37 °C under an atmosphere of 5% CO₂ and selected with 2 µg/mL puromycin for 3 days. The transfection efficiency was confirmed through fluorescence expression.

HAP1 cells were transfected with TurboFectin transfection reagent (OriGene). In brief, 0.8 × 10⁶ cells were seeded in a 6-well plate and transfected on the following day with 1.5 µg of Cas9 expression plasmid (#48137 from Addgene), 1 µg of U6 driven gRNA expression plasmid (Horizon Discovery), 0.5 µg of PCR product encoding the homology donor and 0.2 µg of plasmid encoding a blasticidin resistance gene. 24 h post transfection, cells were treated with 20 µg/ml of blasticidin for 24 h to eliminate untransfected cells. Single clones were obtained by limiting dilution. Positive clones were then identified by PCR screening.
iPS cells cultured in TeSR-E8 and Vitronectin XF (both STEMCELL Technologies) were transfected with the P3 Primary Cell solution using the CM-138 pulse in the 4D-Nucleofector (Lonza). In brief, 1.0 × 10⁶ cells were transfected with 2.5 µg of Cas9 expression plasmid, 2.5 µg of gRNA expression plasmid, 2.5 µg of plasmid encoding the donor and 0.5 µg of plasmid encoding a blasticidin resistance gene. Cells were plated in culture media supplemented with 10 µM ROCKi (Y-27632, Abcam) for 24 h, and then treated with 10 µg/ml of blasticidin for 24 h to eliminate untransfected cells. Recovered colonies were single cell plated after treatment with StemPro Accutase (Thermo Fisher Scientific) and clones were manually picked into 96 wells for screening.