Quantitative PCR

Total RNA was extracted with TRIzol (Thermo Fisher) and was reverse-transcribed into cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo) in accordance with the manufacturer's instructions. qPCR reactions were performed on a LightCycler480-II (Roche) using THUNDERBIRD Probe qPCR Mix (Toyobo). The sequences of primers designed to be compatible with the Roche Universal Probe Library (UPL) are provided in Supplementary Tables 2 and 3. The hydrolysis probe used in the assay was labelled with a fluorescein-based reporter dye (FAM) and a non-fluorescent quencher. Cycling conditions were the following: 95 °C for 15 min (one cycle), 95 °C for 15 s and 60 °C for 1 min (40 cycles). A total of 1–2 μl of cDNA per sample was used for the quantification of endogenous mRNA levels. Expression levels were normalized to Hprt1 or RPL13A.