qPCR Data of iPSC-CF Activation (ACTA2, COL1A1).xlsx

hiPSC-CF were cultured in T25 flasks for 72 hours in Fibroblast Growth Media 3 supplemented with 10 ng/ml FGF2 + 10 µM SB 431542 or 10 ng/ml FGF2 + 10 ng/mL TGF-β1 (R&D Systems, 240-B-002/CF). RNA extraction was performed using the Direct-zol RNA Miniprep Kit (Zymo Research, R2050) according to the manufacturer’s protocol. cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368814) according to the manufacturer’s protocol. RT-qPCR was performed using TaqMan™ Fast Advanced Master Mix (ThermoFisher Scientific, 4444557) following the manufacturer’s protocol and on a QuantStudio™ 5 Real-Time PCR System, 384-well (ThermoFisher Scientific, A28140). RT-qPCR was performed using TaqMan™ Gene Expression Assay (FAM) (ThermoFisher Scientific, 4331182) for ACTA2 (Hs00426835_g1) and COL1A1 (Hs00164004_m1). All reactions were multiplexed and normalised to the housekeeping gene GAPDH using Human GAPD (GAPDH) Endogenous Control (VIC™/MGB probe, primer limited) (ThermoFisher Scientific, 4326317E). 10 ng of cDNA was used per reaction. Transcript levels were measured from 3 samples of iPSC-CF, with each sample being obtained from cells at different passages. Cycle threshold values were obtained from 3 technical replicates of each sample. Relative gene expression was calculated using the comparative cycle threshold method (Schmittgen et al., 2008).