Lioac

Chemically competent yeast cells were created following the lithium acetate protocol (Gietz and Schiestl, 2007 (link)). Yeast colonies were grown to saturation overnight in YPD. The following morning the cells were diluted 1:100 in 15 mL of fresh YPD in a 50 mL conical tube and grown for 4-6 h to OD600 0.8-1.0. Cells were pelleted and washed once with 10 mL 0.1 M lithium acetate (LiOAc) (Sigma). Cells were then resuspended in 0.1 M LiOAc to a total volume of 100 μL/transformation. 100 μL of cell suspension was then distributed into 1.5 mL reaction tubes and pelleted. Cells were resuspended in 64 μL of DNA/salmon sperm DNA mixture (10 μL of boiled salmon sperm DNA (Invitrogen) + DNA + ddH₂O), and then mixed with 294 μL of PEG/LiOAc mixture (260 μL 50% (w/v) PEG-3350 (Sigma) + 36 μL 1 M LiOAc). The yeast transformation mixture was then heat-shocked at 42°C for 40 mins, pelleted, resuspended in 200 μL 5 mM CaCl₂ and plated onto the appropriate synthetic dropout medium.

For transformation into yeast, 200 ng of the final CRISPRai plasmid was digested at 37 °C for 1 h with NotI in the following setup: 200 ng CRISPRai, 1 µL CutSmart Buffer (NEB), 0.2 µL NotI-HF (NEB), up to 10 µL H₂O. Digestions were heat inactivated at 65 °C for 20 min before transformation. Chemically competent yeast cells were transformed using the lithium acetate protocol from Gietz and Schiestl^{40 (link)}, as follows: Yeast colonies were grown to saturation overnight in YPD. The following morning the cells were diluted 1:100 in 15 mL of fresh YPD in a 50 mL conical tube and grown for 4-6 h to OD₆₀₀ 0.8-1.0. Cells were pelleted and washed once with 10 mL 0.1 M lithium acetate (LiOAc) (Sigma). Cells were then resuspended in 0.1 M LiOAc to a total volume of 100 μL/transformation. 100 μL of cell suspension was then distributed into 1.5 mL reaction tubes and pelleted. Cells were resuspended in 64 μL of DNA/salmon sperm DNA mixture (10 μL of boiled salmon sperm DNA (Invitrogen) + DNA + ddH₂O), and then mixed with 294 μL of PEG/LiOAc mixture (260 μL 50% (w/v) PEG-3350 (Sigma) + 36 μL 1 M LiOAc). The yeast transformation mixture was then heat-shocked at 42 °C for 40 mins, pelleted, resuspended in 200 µL sterile H₂O and plated onto the appropriate selection medium.

The NMY51 TOR1-1 Δfpr1 yeast strain was generated as previously described23 (link). For expression of bait and prey combinations of interest, NMY51 TOR1-1 Δfpr1 was transformed according to DUALhunter^TM kit instructions (Dualsystems biotech.). Competent cells were obtained by diluting O/N YPAD grown culture to OD₆₀₀ = 0.2 followed by incubation at 30 °C (120 rpm) for 4–6 hours in YPAD. Cells were harvested (700 × g for 5 min), followed by brief washing in 0.1 M LiOAc (Sigma) and resuspension in 100 μL sterile water per transformation. Mastermix consisting of 600 ng DNA per plasmid (1200 ng total), 240 μL 50% PEG (Sigma), 36 μL 1M LiOAc (Sigma) and 25 μl preboiled ssDNA (2 mg/mL, Sigma) was added, followed by incubation in 42 °C water (45 min). Transformed cells were harvested by centrifugation (700 × g for 5 min), resuspended in 100 μL sterile H₂O and grown at 30 °C for 3 days on synthetic complete dropout media (Sunrise Science Products) with yeast nitrogen base (Duchefa) and glucose, but lacking leucine and tryptophan (SD-LW).