Next-generation sequencing of targeted loci modified by SMART editing
Next-generation sequencing of targeted loci modified by SMART editing
Transfected cells were harvested two to three days after Cas9 RNP transfection. To detach and dissociate the cells, they were treated with trypsin. Cell pellets were then collected by centrifugation. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (cat# 69506). The concentration of genomic DNA ranges from 100 to 200 ng/μl.   The edited regions in target genes were amplified by PCR and sequenced on an Illumina MiSeq. Amplification primers containing partial Illumina forward and reverse adapters were used in the first round of PCR to amplify the edited regions. The first PCR reaction (25 µl) contained 12.5 µl of Q5 High-Fidelity 2X Master Mix (cat# M0492L), 0.5 µM forward and reverse primers, and 150 ng of genomic DNA. The PCR program was as follows: 98°C for 3 minutes; 30 cycles of 98°C for 15 seconds, optimized annealing temperature for 30 seconds, and 72°C for 15 seconds; followed by a final extension at 72°C for 2 minutes. PCR products were analyzed by gel electrophoresis, and concentrations were quantified. In the second PCR, full-length Illumina adapters were added to each sample using NEBNext Multiplex Oligos for Illumina (NEB, cat# E6440S). The second PCR reaction (50 µl) contained 25 µl of Q5 High-Fidelity 2X Master Mix, 0.125 µM index primers (higher concentrations resulted in amplification bias), and 1 ng of first-round PCR products. The program for the second PCR was as follows: 98°C for 1.5 minutes; 12 cycles of 98°C for 15 seconds, 72°C for 30 seconds, and 72°C for 20 seconds; followed by a final extension at 72°C for 2 minutes. PCR products were analyzed by gel electrophoresis and purified using the NucleoSpin Gel and PCR Clean-Up Kit. DNA concentration was measured using Qubit. All samples were pooled at an equimolar ratio and sequenced on the Illumina MiSeq platform.