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Download Corel Draw X6 Full Version 32-bit (Final 2022)



 


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NET Framework 4 or later. Quantitative real-time RT-PCR {#Sec6} ----------------------------- Based on the results of our screen, we performed qRT-PCR on two genes in the screen, *DDA1* and *CUL4A*, and on two genes, *RPT6* and *XRN1*, in the validation. A ratio of *CUL4A* to *β-actin* was calculated for each sample in the screen. A ratio of *RPT6* to *β-actin* was calculated for each sample in the validation. The relative expression of the candidate genes was calculated with the 2^-ΔΔCt^ method and normalized to the endogenous control gene *β-actin*. The sequences of qRT-PCR primers used are listed in Additional file [1](#MOESM1){ref-type="media"}: Table S1. Results {#Sec7} ======= First, we generated a collection of HeLa cell lines stably expressing Cas9 and sgRNAs targeting a reporter construct by targeting the endogenous *β-actin* gene (Additional file [2](#MOESM2){ref-type="media"}: Figure S1A). By this strategy, we obtained a collection of cells with Cas9-mediated insertions at a variety of locations in the genome (Additional file [2](#MOESM2){ref-type="media"}: Figure S1B and C). The cumulative insertion frequency of HeLa cells stably expressing Cas9 and sgRNAs targeting the *β-actin* gene is approximately 0.1 %. However, the cumulative insertion frequency of cell lines with mutations in *DDA1*, *CUL4A*, *RPT6*, and *XRN1* was approximately 1.3 %, significantly higher than that of the *β-actin* cells (Additional file [2](#MOESM2){ref-type="media"}: Figure S1D). As shown in Additional file [2](#MOESM2){ref-type="media"}: Figure S1, the location of the insertions are spread across the human genome. To obtain cells with mutations in the genes of interest, a set of gene-targeting sgRNAs was designed and the corresponding donor constructs were generated by insertion of homology

 

 

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Download Corel Draw X6 Full Version 32-bit (Final 2022)
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