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 protocol70 (link).
All CRISPR/Cas9 experiments were compared to non-targeting control (NTC) obtained with the sgRNAs, listed in Supplementary Table7 , 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.
All CRISPR/Cas9 experiments were compared to non-targeting control (NTC) obtained with the sgRNAs, listed in Supplementary Table
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.
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