Synth a freeze cryopreservation media

To screen for fluorescence, organoids were imaged in 96-well round bottom plates (#650180; Greiner, Frickenhausen, Germany) using an ImageXpress under widefield settings. For RNA-seq, 2 × 10⁵ SIX6-GFP/VSX2-tdTomato undifferentiated control hPSCs (n = 3) and differentiated organoids were collected based on morphology (days 05 to 45), GFP single fluorescence (days 15 to 20) and GFP-tdTomato dual fluorescence (days 25, 35 and 45). Organoid samples were pooled as triplicates (n = 8–20 organoids/replicate days 05 to 25, n = 2–5 organoids/replicate days 35 and 45, washed three times with PBS (Ca²⁺/Mg²⁺ free) and frozen at −80 °C. For ATAC-seq, triplicate samples of SIX6-GFP/VSX2-tdTomato hPSCs (2 × 10⁵ cells; day 00), SIX6-GFP/VSX2-tdTomato (n = 8–20 pooled organoids/replicates for days 5 through 25 and n = 1 organoid/replicate for day 45) were collected in Synth-a-Freeze Cryopreservation Media (#A1254201; Thermo Scientific, Waltham, MA, USA) and stored in liquid nitrogen until preparation (see nuclei isolation below).