For culture experiments, cells were grown at
4%O2,7% CO2 and serum-starved 24h before
harvesting. mRNA was isolated using Qiazol (Qiagen) and Direct-zol RNA Mini Prep
(R2052, Zymo Research), or Absolutely RNA Nanoprep Kit (400753, Agilent
Technologies). Samples were quantified using a Nanodrop spectrophotometer
(Thermo Scientific). cDNA was synthesized from 300 ng to 1.5 µg of total
RNA using SuperScript VILO with random primers (Invitrogen) or SuperScript III
First-Strand Synthesis System (Invitrogen) with oligo(dT) primers. Quantitative
real-time PCR (qRT-PCR) was performed with Absolute Blue QPCR SYBR Green Mix
plus ROX Vial Kit (AB-4166, Thermo Scientific) on a MyiQ2 (BioRad), or with
LightCycler DNA Master SYBR Green I reagents (Roche) on a Light Cycler 480
(Roche). Measurements were recorded in duplicate or triplicate. Differences
between samples and controls were calculated based on the
2−ΔΔCT method and normalized to Rplp0
(mouse) or RPLP0 (human). For detailed list of primer sequences seeSupplementary Table
3 .
4%O2,7% CO2 and serum-starved 24h before
harvesting. mRNA was isolated using Qiazol (Qiagen) and Direct-zol RNA Mini Prep
(R2052, Zymo Research), or Absolutely RNA Nanoprep Kit (400753, Agilent
Technologies). Samples were quantified using a Nanodrop spectrophotometer
(Thermo Scientific). cDNA was synthesized from 300 ng to 1.5 µg of total
RNA using SuperScript VILO with random primers (Invitrogen) or SuperScript III
First-Strand Synthesis System (Invitrogen) with oligo(dT) primers. Quantitative
real-time PCR (qRT-PCR) was performed with Absolute Blue QPCR SYBR Green Mix
plus ROX Vial Kit (AB-4166, Thermo Scientific) on a MyiQ2 (BioRad), or with
LightCycler DNA Master SYBR Green I reagents (Roche) on a Light Cycler 480
(Roche). Measurements were recorded in duplicate or triplicate. Differences
between samples and controls were calculated based on the
2−ΔΔCT method and normalized to Rplp0
(mouse) or RPLP0 (human). For detailed list of primer sequences see
3