Quantifying Gene Expression via qPCR

RNA extraction, reverse transcription, and quantitative PCR (qPCR) was generally performed as described previously.¹⁰³ (link) Cultured cells were directly lysed in the culture plate, or alternatively, were purified via FACS and then lysed, using 350 μL of RLT Plus Buffer. RNA was then extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol. 300 ng of total RNA was reverse transcribed into cDNA for qPCR using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed in 384-well format as previously described,¹⁰³ (link) using gene-specific forward and reverse primers, as well as the SensiFAST SYBR Green Lo-ROX Kit (Thomas Scientific), on a QuantStudio 5 qPCR machine (Thermo Fisher). qPCR primer sequences are provided in Table S4. Expression of all genes was normalized to the levels of the reference gene YWHAZ.