Cell dissociation for single-cell RNA sequencing and flow cytometry analysis

Day69 control (WNTi) and treated (WNTi + FGF8) organoids were dissociated using an enzymatic Papain Dissociation System (Serlabo technologies LK003150 or Worthington, #LK003150), by following manufacturer’s instructions. Low-bind Eppendorf tubes were used to limit material loss. Two to three organoids per batch were isolated in Eppendorf tubes containing 1 ml of pre-warmed Papain solution supplemented with DNase. Total incubation lasted 70 min, with manipulations every ten minutes that consisted in gentle mixing by tube inversion (first 2 rounds) then gentle pipetting (following rounds), ending up with addition of Ovomucoid solution for Papain inactivation. Samples were filtered (Cell Strainer 40 µm Nylon, FALCON, 352340) to remove undissociated cells, spinned down (1200 rpm, corresponding to 170 g, for 5 minutes) and resuspended in PBS1x supplemented with 0.1% BSA (Jackson, 001-000-161). Cell viability upon dissociation was checked by Propidium Iodide (PI) staining in flow cytometry in an independent experiment, where Papain was compared with Accutase and with ReLeSR (Stem Cell Technologies, #100–0483). For flow cytometry, cells were stained with Propidium Iodide (40 µg/ML, Sigma, P4170) in PBS1x for 15 min at RT, washed twice with PBS1x supplemented with 1% Serum, then analysed with BD LSRFortessa and FACSDiva software (Becton Dickinson) to measure dying cells which incorporated PI. Cells were analysed on the basis of 10,000 total events (debris excluded). Cell viability was tested again by Trypan blue staining just prior cell counting for library preparation, and samples were considered suitable for single cell RNA sequencing only when damaged cells detected by Trypan blue or by PI staining were ≤10% of the total population.