2.3. Isolation of Total RNA from Organoid Cultures

For isolation of RNA from organoid cultures, matrigel domes were resuspended in 1 mL ice-cold 1× PBS. The organoids were centrifuged at 5500 rpm and 4 °C for 6 min. The organoid pellets were resuspended in 500 mL Cell Recovery Solution and incubated on ice for 60 min. Following, the organoids were centrifuged (same settings as before) and washed once with 500 µL ice-cold 1× PBS. The supernatant was discarded and the organoid pellets were immediately frozen at −80 °C. RNA was isolated using the RNeasy^® Plus Mini Kit (Qiagen, Hilden, Germany) and QIAshredder (Qiagen) according to manufacturer’s instructions. Briefly, organoid pellets were resuspended in 350 µL Buffer RLT Plus, loaded on a QIAshredder column and centrifuged at 13,000 rpm for 2 min. The homogenate was transferred to a gDNA Eliminator spin column in a new collection tube and centrifuged for 30 s at 10,000 rpm. A total volume of 525 µL 100% ethanol was added to the flow-through and shortly vortexed. 700 µL of the mixture were loaded onto an RNeasy Mini spin column and centrifuged at 10,000 rpm for 30 s. The flow-through was discarded and the step was repeated until the whole sample passed the membrane. Following, 500 µL Buffer RPE was added to the spin column and centrifuged at 10,000 rpm for 30 s. The flow-through was discarded and the step was repeated once. The mini spin column was centrifuged at 13,000 rpm for 2 min to dry the membrane. The column was placed in a new collection tube, 35 µL RNase-free water were added onto the membrane and columns were incubated at RT for 3 min. Following, samples were centrifuged at 13,000 rpm for 2 min to elute total RNA.