Viia real time pcr

The extent of DNA 5′ end resection was analyzed by RT-qPCR analysis of the DNA surrounding the I-SceI site of the GFP gene integrated in the U2OS cells (U2OS DR-GFP reporter) transfected with the I-SceI expression plasmid. Primers flanking the I-SceI site and four regions located approximately 250 bp, 300 bp, 350 bp and 2200 bp away from this site were used. The housekeeping gene GAPDH was used as a genomic DNA control. RT-qPCR was performed in the ViiA Real Time PCR (Applied Biosystems, Foster City, CA) using Power SYBR Green PCR Master Mix (Applied Biosystems). The percentage of digestion was estimated as follows: 2^(Ct reference–Ct sample), where Ct reference and Ct sample are mean threshold cycles of RT-qPCR done in duplicate on DNA samples from GAPDH (reference) and the DNA regions surrounding the I-SceI site (sample). The amount of DNA was corrected for the I-SceI digestion efficiency by normalizing the data against the amount of DNA flanking the restriction site. All primer sequences are presented in Table 1.

Total RNA was extracted with TRIzol (Invitrogen). cDNA was made using Superscript II Reverse Transcriptase (Invitrogen). RT-qPCR was performed in the ViiA Real Time PCR (Applied Biosystems, CA, USA), using SYBR Green PCR master mix (Applied Biosystems). The relative RNA expression was estimated as follows: 2^(Ct reference – Ct sample), where Ct reference and Ct sample are mean threshold cycles of RT-qPCR done in duplicate on cDNA samples from U6 snRNA (reference) and the cDNA from the genes of interest (sample). All primer sequences are presented in Supplementary file 1I.