*2.8. RNA Preparation and Quantitative RT-PCR (RT-qPCR)*

Total RNA was isolated using a combined TRI reagent and silica column protocol. Per 24 well, cells were lysed in 500 μL TRI reagent (TriFast, VWR, Darmstadt, Germany) and incubated for 5 min at RT. The lysate was transferred into a 1.5 mL tube and 200 μL chloroform was added, mixed well by shaking and incubated for 5 min at RT. The samples were centrifuged at 12,000 g for 15 min and the upper aqueous phase (~300 μL) was pipetted into a new 1.5 mL tube containing 300 μL 100% ethanol. After mixing, the sample was loaded onto the column (Zymo-Spin IC, Zymo Research, Freiburg, Germany), prior to centrifugation at 8000 g for 1 min. The flow-through was discarded. The column was washed twice with 450 μL 3 M sodium acetate pH 5.2, and once with 70% ethanol by centrifugation at 8000 g for 1 min; the collection tube was emptied after each step. For the drying step, the collection tube was replaced by a new one, and the columns were centrifuged at 12,000 g for 2 min. For RNA elution, 15 μL DEPC-water, pre-warmed to 65 ◦C, was added onto the column and incubated for 2 min, before finally eluting the RNA by centrifugation at 8000 g for 2 min. RNA concentrations and purities were measured in a NanoDrop 2000c (Thermo Fisher, Schwerte, Germany).

The total RNA was reverse transcribed with the RevertAid™ H Minus First strand cDNA synthesis Kit (Thermo Fisher, Waltham, MA, USA). In brief, 1 μL random hexamer primer was added to 1 μg RNA diluted in 10 μL DEPC-treated water, followed by an incubation step for 5 min at 65 ◦C and cooling to 4 ◦C. Then, 4 μL 5× reaction buffer, 2 μL 10 mM dNTP mix, 2.5 μL DEPC-treated ddH2O, and 0.5 μL RevertAid™ H Minus M-MuLV Reverse Transcriptase (200 U/μL) were added. The cDNA synthesis mixture was incubated at 25 ◦C for 10 min, 42 ◦C for 60 min, and heat denatured at 70 ◦C for 10 min.

For quantitative real time PCR, a StepOnePLUS Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used, applying the ΔΔCt method [54]. The cDNA was 1:10 diluted with DEPC-water and 4 μL were mixed with 5 μL 2x PerfeCTa SYBR® Green FastMix ROX (Quantabio, Beverly, MA, USA) and 1 μL 5 μM forward and reverse primer mix (see Table S2 for primer sequences). The qPCR were run with a pre-incubation at 95 ◦C for 2 min, followed by 45 amplification cycles (95 ◦C for 15 s, 55 ◦C for 15 s, 72 ◦C 15 s).
