For each primer pair, we validated the quantification and specificity prior to use. Specifically, we tested primers in qPCR reactions using a dilution series of mouse ES cDNA including a no reverse transcriptase (RT) sample. We excluded any primer that did not have robust quantification across a 64-fold dilution curve, had high signal in the no RT sample, or had low detectable expression in the undiluted sample (cycle number >34). For primers that failed this validation we redesigned and tested new primers.
