Bravo automated liquid handling platform

Single cells and clumps were sorted with SORP-FACSAriaII machine with BD FACSDIva^TM software (BD Biosciences) using a 100 μm nozzle. For clumps sorting, dead cells were excluded using the Zombie green staining and clumps were sorted based on Hoechst histogram (Fig. 1b, Supplementary Fig. 18a). For single cell sorting, dead cells were excluded on the basis of 500 ng/ml Dapi incorporation. Sorted cells were negative for CD45 and positive for Epcam (Supplementary Fig. 18b). To enrich for enteroendocrine cells, cells were gated on CD45^- Epcam⁺ CD24⁺. Since tuft cells express CD45^{17 (link)}, to enrich for those, cells were gated only on Epcam⁺ CD24⁺ (Supplementary Fig. 18c).
Cells and clumps were sorted into 384-well MARS-seq cell capture plates containing 2 μl of lysis solution and barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq. Barcoded single cell capture plates were prepared with a Bravo automated liquid handling platform (Agilent) as described previously^{16 (link)}. Four empty wells were kept in each 384-well plate as a no-cell control during for data analysis. Immediately after sorting, each plate was spun down to ensure cell immersion into the lysis solution, snap frozen on dry ice and stored at −80 °C until processed.

2,500 293A-TEAD-LUC cells were plated in 50 μL of growth medium without FBS in white 384-well plates (Corning) and allowed to proliferate for 48 h until fully confluent. 100 nL of compound solvated in DMSO was then transferred to each well using a Bravo Automated Liquid Handling Platform (Agilent) affixed with a pintool head (V&P Scientific). For TEAD-LUC activity assays, 30 μL of BrightGlo reagent solution (Promega, diluted 1:3 in water) was added to each well after 24 h of compound treatment. For cytotoxicity assays, 30 μL of CellTiter-Glo reagent solution (Promega, diluted 1:6 in water) was added to each well after 24 h and 72 h of compound treatment. Luminance values were recorded on an Envision plate reader (Perkin Elmer).

Bilayer formation and protein deposition was performed as previously described[19 (link)] except for the following changes. A custom 384 well microtiter plate (Biodesy Delta Read Plates) was used to form supported lipid bilayers for protein deposition. A Bravo automated liquid-handling platform (Agilent) was used for bilayer deposition, buffer washes, and protein deposition during sample preparation.
For MLKL experiments, the assay buffer was 20mM HEPES, pH 8.0, 200mM NaCl, 0.005% Tween-20. MLKL protein was deposited at a final concentration of 1μM in each well and allowed to incubate on the Ni-NTA surface overnight at 4°C. After overnight incubation, unbound protein was removed by washing with assay buffer plus 1% DMSO. Plates were then allowed to equilibrate for 30 minutes at room temperature before beginning compound injections.

Cells (5000/well) were seeded in duplicate in a 384-well culture plate in RPMI-1640 medium containing 10% FBS and cultured overnight at 37 °C with 5% CO₂. Ten micromolar of 119 FDA-approved anti-cancer drugs (Selleck Chemicals) were added to the cells, using the Bravo Automated liquid handling platform (Agilent technologies), and cell viability was assessed 72 h later with a CCK-8 kit.
To determine the cell sensitivity to the drugs, serially diluted anti-cancer drugs in RPMI-1640 medium containing 10% FBS were added to cells and cultured for 72 h. Cell viability was then assessed by CCK-8. IC₅₀ values were calculated by drawing four-parameter curve fitting using GraphPad Prism (version 7, Graphpad Software). The study was performed in duplicate.