Cells were harvested using Accutase, quenched in DMEM/F12, and spun down for 5 min at 200g. After removing the supernatant, cell pellets were snap-frozen at −80°C until ready for mRNA extraction. The RNeasy Mini Kit (Qiagen) was used to extract mRNA, including a cell lysate homogenization step on QIAshredder columns (Qiagen), according to manufacturer instructions. DNA was removed on column using the RNase-free DNase Set (Qiagen). Extracted RNA was stored in nuclease-free water at −20°C until ready to reverse transcribe to complementary DNA (cDNA). RNA was reverse-transcribed at a concentration of 250 ng/ml into cDNA using an OmniScript Reverse Transcriptase Kit (Qiagen) and Oligo(dT)20 Primers (Life Technologies). Temporal gene expression analysis was conducted using 25-μl reactions containing GoTaq Green Master Mix (Promega), 10 ng of cDNA template per reaction, and 100 nM forward/reverse primers. PCR was run according to manufacturer protocols, and all reactions included a no template and no reverse transcription control to verify the absence of genomic DNA contamination or amplification. PCR primer sequences, annealing temperatures, and cycle times are listed in table S1B. PCR products were resolved on a 2% agarose gel, stained using ethidium bromide, and imaged on a ChemiDoc XRS+ System (Bio-Rad).

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