Superscript 4 synthesis kit

Total RNA was extracted from aiFACS-sorted cells using TRIzol reagent (Sigma-Aldrich) according to the manufacturer's instructions. In each experimental sample, 1 μg of RNA (Supplemental Fig. S2) or 5000 cells (Figs. 2B, 4B, 5A,B; Supplemental Fig. S11) were used for each condition. RNA was purified using 500 μL of TRIzol reagent (Sigma-Aldrich) and precipitated from the aqueous phase with 500 μL of isopropanol (VWR Medicals) and 1 μL of glycogen (20 μg/μL, Invitrogen). RNA was resuspended in 20 μL (Supplemental Fig. S2) or 11 μL (Figs. 2B, 4B, 5A,B; Supplemental Fig. S11) of nuclease-free H₂O. Either 1 μg (Supplemental Fig. S2) or 11 μL (Figs. 2B, 4B, 5A,B; Supplemental Fig. S11) of RNA was added to the RT reaction that was performed using the SuperScript IV synthesis kit (Invitrogen). Initial amplification was performed with a denaturation step for 5 min at 65°C, followed by oligo(dT) annealing for 10 min at 23°C, primer annealing for 10 min at 53°C, and primer extension for 10 min at 80°C. Upon completion of the cycling steps, the reactions were stored at −20°C. Quantitative PCR (RT-qPCR) was performed on a light cycler 480 (Roche) with MasterMix SYBR Green (Roche) following the manufacturer's instructions and according to the MIQE guidelines (Bustin et al. 2009 (link)). Primer sequences are listed in Supplemental Table S1.