Dmem f12

Isolated brains from Smarcb1^{1148del/1148del} and Smarcb1^+/+ P0 mice were divided into a supratentorial and an infratentorial part and each was minced with scalpels on ice. For enzymatic dissociation, 2–3 mL of a solution containing 1vial papain (Worthington, #LK003178), 5 mL pre-equilibrated DMEM:F12 (Gibco, #11330-032) and 340 µg DNAse I (Worthington, #2139) diluted in 250 µL DMEM:F12 were added to each sample, prior to incubation at 37 °C in 5%CO₂ for 30 min. The samples were gently shaken every 3–5 min and finally transferred with 1 mL wide bore tips (Starlab, #E1011-9000) onto a 40 μm cell strainer (Corning, #352340). The reaction was stopped with 2 mL PBS/10%BSA, followed by washing with 10 mL PBS. The samples were centrifuged at 1200 rpm for 8 min at room temperature. The pellet was then resuspended in neural stem cell (NSC) medium, containing DMEM:F12 (Gibco, #11330-032), 100 U/mL penicillin–streptomycin (Gibco, #15140-122), 1 × B-27™ supplement minus vitamin A (Thermo Fisher Scientific, #12587-010), 20 ng/mL recombinant murine EGF (Peprotech, #315-09) and 20 ng/mL recombinant murine FGF-basic (Peprotech, #450-33), for a subsequent scRNA-seq procedure or cell culture.

Single-cell suspensions of endolymphatic sac epithelia were prepared from E12.5, E16.5, P5 or P30 C57BL/6J mice of both sexes for RNA-seq or qPCR analysis. Inner ears were removed from four to five mice and placed in ice-cold DMEM/F12 (GIBCO). The endolymphatic sac was harvested together with surrounding connective and osseous tissues. The tissues were pooled and incubated in 1 mg/ml collagenase (LS004194, Worthington, Lakewood, NJ) and 1 mg/ml dispase (LS02100, Worthington) in DMEM/F12 at 37°C for 10 min. The tissues were then transferred into 10% fetal bovine serum (FBS) (GIBCO) in DMEM/F12 where the epithelia were isolated from surrounding tissues. The epithelia were transferred to 50 µl of 0.125% trypsin/EDTA (GIBCO) in phosphate-buffered saline (PBS) in a 1.5-mL tube and incubated for 15 min at 37°C. Then 50 µL of 10% FBS in DMEM/F12 was added to the tube to stop digestion. The epithelia were gently triturated with a 200-µl micropipette tip to complete the dissociation. Cell concentration was measured using Luna-FL automated cell counter (Logos Biosystems, Annandale, VA) and adjusted to 250,000 to 300,000 cells/ml by adding 5% FBS in DMEM/F12. To assess cell viability after cell capturing, the cells were labeled with 1:1000 of calcein-AM or ethidium homodimer-1 (EthD-1) (LIVE/DEAD cell viability assay, Thermo Fisher Scientific, Waltham, MA) and placed on ice until capture.

The healthy adult pancreatic ductal cells (PDCs) were cultured as previously described^{34 (link)}. Briefly, cells were seeded on collagen coated plates (a 3 mL collagen type I layer (2.31 mg/mL) on a tissue culture dish), and were grown in PDC medium: DMEM/F-12 (Thermo Fisher Scientific), 5 mg/mL D-glucose (Sigma Aldrich), 0.5% ITS premix (Corning), 5% Nu-Serum (Corning), 1x Penicillin/Streptomycin (Thermo Fisher Scientific), 25 µg/mL Bovine Pituitary Extract (Thermo Fisher Scientific), 100 ng/mL Cholera Toxin (Sigma Aldrich), 1 µM Dexamethasone (Sigma Aldrich), 10 mM Nicotinamide (Sigma Aldrich), 100 µg/mL Primocin (Invivogen) and 20 ng EGF (R & D systems) (Table S1). Media changes were performed every 48 h and upon 80–85% confluency the collagen was further digested for 15 min at 37 °C with 1.5 mg/mL Collagenase Type 4 (Worthington) diluted in DMEM/F-12, then cold PBS was added and the mixture was centrifuged. The cell pellet was then trypsinized and Soybean Trypsin Inhibitor (STI) was used to quench the effect of trypsin. Afterwards, 10.000 cells were seeded into collagen gels (as described in “Organoid preparation”). The procedure is schematized in Fig. S7b.