Ezna fastfilter endo free plasmid dna maxi kit

To knock-out Zeb1, Hdac2, and eNOS the target-specific sgRNAs, listed in Supplementary Table 7, were cloned into LentiCRISPR2 vector (addgene, Cambridge, MA) using the GoldenGate protocol^{70 (link)}.
All CRISPR/Cas9 experiments were compared to non-targeting control (NTC) obtained with the sgRNAs, listed in Supplementary Table 7, cloned into the LentiCRISPR2 vector using the GoldenGate protocol.
The obtained plasmids were transformed into NEB 5-alpha Competent Escherichia coli (High Efficiency –New England Biolabs), then DNA was purified by EZNA Fastfilter Endo-Free Plasmid DNA Maxi Kit (Omega Bio-Tek), and a concentration of 6 μg was used for nucleofection. Nucleofection was performed in 10⁶ mESC cultured in GS using Amaxa P3 primary cell 4D Nucleofector Kit (Lonza). After 48 h, nucleofected mESC were selected by 1.5 μg mL⁻¹ puromycin. After recovery from selection, mESC were tested for Zeb1, Hdac2, and eNOS knockout by western blot. mESC nucleofected with eNOS CRISPR/Cas9 vectors resulted knocked out and were used for subsequent experiments, whereas mESC nucleofected either with Zeb1 CRISPR/Cas9 vectors or Hdac2 CRISPR/Cas9 vectors were clonally expanded. Once monoclonal Zeb1_1 and Zeb1_2 CRISPR/Cas9 mESC as well as monoclonal Hdac2_1 and Hdac2_2_CRISPR/Cas9 mESC were obtained, expression analysis of mesendodermal markers was conducted.

To inactivate mouse KGDH, sgRNAs were cloned into LentiCRISPR2 vector (Addgene) using the GoldenGate protocol:^{68 (link)}- KGDH_1: Fw 5′-caccgCAGCATCCAAAATCCCCAG-3′;
Rv 5′-aaacCTGGGGATTTTGGATGCTGc-3′;
- KGDH_2: Fw 5′-caccgGTGAACTGCATGATCCCAG-3′;
Rv 5′-aaacCTGGGATCATGCAGTTCACc-3′;
Specific CRISPR/Cas9 transfection was compared to a non-targeting control (NTC) sgRNA:
- NTC: Fw 5′-caccgTTCCGGGCTAACAAGTCCT-3′;
Rv 5′-aaacAGGACTTGTTAGCCCGGAAc-3′.
The obtained plasmids were transformed into NEB 5-alpha Competent E. coli (High Efficiency –New England Biolabs), then DNA was purified by EZNA Fastfilter Endo-Free Plasmid DNA Maxi Kit (Omega Bio-Tek), and a concentration of 6 μg was used for transfection in 4T1 cells; the transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. All experiments were performed by using the polyclonal population emerging after a round of puromycin selection showing residual KGDH expression, specifically, after 48 h 4T1 cells were selected by 1.5 μg/mL puromycin recovered from selection and tested for KGDH knock-out by western blot.