Itaq sybr green premix

Total RNA was isolated using RNeasy mini kit (Qiagen) column purification. cDNA was synthesized with iScript kit (BioRad). Real-time PCR was performed with iTaq SYBR Green premix (BioRad) and data were collected with LightCycler 480 (Roche). All Ct values were normalized to the expression values of GAPDH RNA and gene expression quantification were performed by the 2^-ΔΔCt method [73 (link)]. The primers sequences are provided in S6 Table.

Equal numbers of WT or OTUB1 CRISPR knockout U2OS cells were plated, and the next day were transfected with either control siRNA or OTUB1 SMARTpool siRNA with Lipofectamine RNAimax. Two days after transfection, cells were harvested and total RNA was extracted with a GenElute kit (Sigma), following the manufacturer's protocol. cDNA was synthesized from 200 ng of total RNA using the NEB ProtoScript First Strand cDNA synthesis kit, following the manufacturer's protocol. RT-PCR contained 1 μl of cDNA, 500 nm forward and reverse primers, and iTaq SYBR Green premix (Bio-Rad). Reactions were run on a QuantStudio RT-PCR machine (Thermo Fisher). Relative mRNA values were quantified from ΔΔC_t values extracted for UBE2E1 and OTUB1 genes compared with TBP using 3 experimental and 2 technical replicates. Primers used for RT-PCR were: 5′-AGATGTTATCGCCTTTGGGA-3′ and 5′-TCCAAACTCCTCTCCACCAG-3′ for UBE2E1; 5′-GCCATAAGGCATCATTGGAC-3′ and 5′-AACAACAGCCTGCCACCTTA-3′ for TBP; 5′- AACACGTTCATGGACCTGATTG-3′ and 5′-TGCTCTGGTCATTGAAGGAGG-3′ for OTUB1 primer set 1; and 5′-CCATCATGGCTCAGCAGGA-3′ and 5′-GAGGTCCTTGATCTTCTGTTGATAGATG-3′ for OTUB1 primer set 2.

Following RNA extraction, sample cDNA was synthesized with the iScript kit (BioRad). Quantitative real time PCR was performed with iTaq SYBR Green premix (BioRad) in 96-well plate format, with data collection and analysis conducted on a LightCycler 480 (Roche). All Ct values were normalized against GAPDH expression. Quantification of gene expression was calculated by the ΔΔCt method, as previously described [85 (link)]. Primer sequences are provided in Table S5.