Single-cell isolation and sorting for RNA-seq

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.