RNA isolation and PCR-based expression analysis

RNA was isolated from sorted cells using the RNeasy Mini Kit (Qiagen). Contaminating DNA was eliminated through on-column DNase digestion (Zymo Research). To generate complementary DNA (cDNA) for RT-PCR analysis, we used the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR amplification and quantification were performed using a real-time PCR machine (QuantStudio 5, Thermo Fisher). Amplification was done using the LightCycler FastStart DNA Master SYBR Green I Kit (Roche), and Hprt was used as housekeeping gene. Reactions were performed in duplicate, and relative gene expression was calculated using the ΔΔC_t method.