Quantitative Real-Time PCR Protocol

1 x 10⁸ cells were harvested at 800 x g for 10 min at 4°C and washed with ice-cold PBS and quick-frozen in dry ice for 1 min. RNA was purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was quantified using an ND-1000 spectrophotometer and Nanodrop software (Nanodrop Technologies). cDNA synthesis and qRT-PCR reaction setup was performed as described previously [56 (link)]. qRT-PCR was performed using iQ-SYBRGreen Supermix on a MiniOpticon Real-Time PCR Detection System (Bio-Rad) and was quantified using Bio-Rad CFX Manager software (Bio-Rad). The following primers were used for qRT-PCR: bTub-RTF (5’-CAAGATGGCTGTCACCTTCA-3’), bTub-RTR (5’-GCCAGTGTACCAGTGCAAGA-3’); USP1 RTF (5’-GAGATGGCACCATCACTCCT-3’), USP1 RTR (5’-GTGGGCAGCACCTCTAGAAC-3’); VDU1 RTF (5’-GTCGAAAGACGTGTGGGTTT-3’), VDU1 RTR (5’-GGAGCGAGGGAAGAGAGATT-3’); ISG65-RTF (5’-GAGCATGTTGATAGAGGGATTG-3’), ISG65-RTR (5’-CATTGCTGTTCTCTGATGTCTG-3’); ISG75-RTF (5’-GAGGGCAGCGAGGCCAAG-3’), ISG75-RTR (5’-CTTCCTACGGCCCCTAATAAC-3’).