Expression of target genes in islet cells was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). RNA was extracted from intact islets and spheroids using RNeasy Plus Mini Kits (Qiagen, Hilden, Germany) and synthesized to complementary DNA (cDNA) by reverse transcription using the PrimeScript 1st Strand cDNA Synthesis Kit (TAKARA, Japan) according to the manufacturer’s instructions. qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) in the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). The primer sequences are listed in table S1. Gene expression levels, normalized to housekeeping gene 18S ribosomal RNA (rRNA), were determined relative to intact islets cultured for 7 days.

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