Iq sybr green supermix kit

Chromosome copy number was determined by qPCR ploidy assays according to the protocol of Pavelka et al.^{60 (link)} as described previously^{18 (link)}. Briefly, DNA samples were prepared by the method described above. The oligonucleotide sequences used for qPCR are listed in Supplementary Table 3. Triplicate qPCR reactions were performed using the iQ SYBR Green Supermix kit in a Roche LightCycler 480 instrument, and the qPCR results were analyzed by the modified Ct method^{59 (link)}. Ct values for each chromosome were normalized to the median Ct value for each cell, and then ∆Ct was represented as relative ratio to WT control. For screening assays in Supplementary Fig. 6, each reaction was performed in technical duplicate.

Total RNAs were extracted from LCTCs and BCTCs, using TRIzol RNA Isolation Reagents as per the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNAs were reverse‐transcribed by oligo(dT) primer using Superscript RT‐PCR kit from Roche, according to the manufacturer's instructions. PCR was performed under the following conditions: 940 °C for 3 min; 94 °C for 30 s; 58 °C for 30 s; 72 °C for 30 s for 40 cycles; and 72 °C for 10 min, using IQ SYBR Green Supermix Kit from Roche. Results were analyzed by the relative quantification method and expressed as relative RNA levels (∆CT, difference of cycling threshold). ∆CT values represent CT [gene]‐CT [GAPDH]; thus, higher values indicate relatively lower expression levels.