Bravo pipetting robot instrument

RNA was isolated using the RNeasy Microkit (Qiagen) and reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific), with 1 μg of extracted RNA. The cDNA (1 μL) was then mixed with the relevant primers (4 μL; Integrated DNA Technologies) and LightCycler 480 SYBR Green I Master (5 μL, Roche) using a Bravo pipetting robot instrument (Agilent) and analyzed by quantitative RT-PCR on a LightCycler 480 II instrument (Roche) using a 40× cycle two-step protocol with a 60°C, 1-min annealing/elongation step and a 95°C, and a 30-s denaturation step. The average cycle threshold (CT) values were calculated from three technical replicates and were used to determine the relative gene expression using the ΔΔCT method. The average fold change was based on two different housekeeping genes (ACTB and GAPDH) and the relative gene expression is described relative to undifferentiated hESCs.

Total RNAs were isolated on day of analysis from three individual organoids/sample using the RNeasy Micro Kit (Qiagen, 74004), according to manufacturer instructions. Reverse transcription was performed using Maxima First Strand cDNA Synthesis kit for qRT-PCR (Thermo Fisher, K1641). cDNA was then mixed with the relevant primers (0.95 μM; Integrated DNA Technologies, see Supplementary Table S2) and SYBR Green Master mix (Roche) using a Bravo pipetting robot instrument (Agilent). The analysis was performed with a LightCycler 480 II instrument (Roche) using a 40x cycle two-step protocol with a 60°C, 1 min annealing/elongation step and a 95°C, 30 s denaturation step. The average CT values were calculated from three technical replicates and were used to determine the relative gene expression using the ΔΔCT method. The average fold change was normalized against two different housekeeping genes (ACTB and GAPDH) and the results were given as relative gene expression over undifferentiated hPSCs.