Quantitative Gene Expression Analysis

RNA for gene expression analysis from EndoC-βH1 was extracted using TRIzol reagent (Invitrogen) and synthesized into complementary DNA using the GoScript Reverse Transcriptase System (Promega). RNA for gene expression analysis from primary human pseudoislets was extracted using the PicoPure RNA isolation kit (Life Technologies), and cDNA was synthesized using the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Quantitative qPCR (qPCR) was performed using TaqMan real-time PCR assays on a 7900HT Fast Real-Time PCR System (all Applied Biosystems, Supplementary Table 4). Ct-values were analyzed using the ΔΔCt method, and target genes were normalized to the combined average of the housekeeping PPIA, GAPDH and TBP. CALCOCO2 expression in EndoC-βH1 and primary islets was extracted from previously published and analyzed RNA-seq data^{71 (link),72 (link)}.