Taqman fast advanced master mix solution

Transcript levels were determined by quantitative (q) RT-PCR. To do so, total RNA was extracted from organoids using RNeasy Mini Kit (70104, Qiagen) and reverse transcribed using High-Capacity RNA to cDNA kit (4387406, Thermo Fisher Scientific). Quantitative PCR reaction was performed using 5 ng of cDNA and the Taqman Fast Advanced Master Mix solution (4444557, Thermo Fisher Scientific). The samples were loaded on a MicroAmp EnduraPlate (4483285, Life and technologies) and run on the QuantStudio 7 Flex Real-Time PCR System instrument (Thermo Fisher Scientific). The probes used are given in Supplementary Table S1. Expression values were normalized to GAPDH as internal control.

Total RNA of CHK-Q cell suspension, monolayer on collagen-coated dish (2.0 × 10³ inoculated-cells/cm², day 3), collagen gel sandwich (0.33, 1.7, and 3.5 × 10⁵ inoculated-cells/cm², day 3 and day 7), and organ-derived ECM gel sandwich (1.7 × 10⁵ inoculated-cells/cm², day 3 and day 7) were extracted using spin columns (NucleoSpin RNA; Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. cDNA was synthesized from total RNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Samples were stored at −30 °C until PCR processing.
The QuantStudio™3 Real-Time PCR System and TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Table S2) were used for PCR. The reaction mixture contained 1 μL cDNA sample, 5 μL TaqMan Fast Advanced Master Mix solution, and 4 μL nuclease-free water in a plate predispensed with the TaqMan Gene Expression Assay probes (Thermo Fisher Scientific Inc., Waltham, MA, USA). Forty amplification cycles consisted of 1 s at 95 °C and 20 s at 60 °C. The comparative cycle time (ΔΔCT) method was used to quantify gene expression levels. Expression levels were normalized to Gapdh, and CHK-Q cell suspension was the control condition.