Power sybr green mix

Total RNA was extracted from cells grown in the same condition as for protein extraction. For the reverse transcription, total RNA was treated with DNase I (New England Biolabs), retro-transcribed with M-MuLV Reverse Transcriptase (New England Biolabs) with oligo (dT)₂₀ primer (Euroclone) and murine RNase inhibitor (New England Biolabs). qPCR on retro-transcribed SYM1 and, as reference, ACT1 was performed by using Power Sybr Green mix with ROX (Kapa Biosystems), supplemented with primers qSYM1Fw and qSYM1Rv or ACT1qFw and ACT1qRv, at a final concentration of 120 nM in the AB 7300 (Life Technologies) instrument at default settings: 50°C 2 min, 95°C 10 min, 41 cycles at 95°C for 15 s and 60°C for 1 min, and one cycle at 95°C for 15 s and at 60°C for 15 s. Statistical analysis was performed through an unpaired two-tailed t-test.

Total RNA was extracted from cells grown to early stationary phase in SC medium supplemented with 2% galactose or 0.6% glucose. For the reverse transcription, total RNA was treated with DNase I (New England Biolabs, Ipswich, MA, USA), retro-transcribed with M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) with oligo (dT)20 primer (Euroclone, Milan, Italy) and murine RNase inhibitor (New England Biolabs, Ipswich, MA, USA). qPCR on retro-transcribed ACS2, HFA1, FET3, FTR1, FIT3 and, as a reference, ACT1 mRNA was performed by using Power Sybr Green mix with ROX Reference Dye (Kapa Biosystems, Darmstadt, Germany), supplemented with appropriate primers (Supplementary Table S1) at a final concentration of 120 nM in the AB 7300 (Applied Biosystems, Foster City, CA, USA) instrument at default settings: 50 °C for 2 min, 95 °C for 10 min, 41 cycles at 95 °C for 15 s and 60 °C for 1 min, and one cycle at 95 °C for 15 s and at 60 °C for 15 s.