Blood genomic dna extraction mini kit

The HeLa cervical carcinoma cell line (JCRB9004) was obtained from the JCRB Cell Bank and used as a control. Huh7.5 cells (Blight et al., 2002 (link)) were kindly provided from Dr. Charles M. Rice. Huh7.5.1 cells (Zhong et al., 2005 (link)) were kindly provided from Dr. Francis V. Chisari. Genomic DNA was prepared from the cell lines using the Blood Genomic DNA Extraction Mini Kit (Favorgen, Ping-Tung, Taiwan, ROC). Deletions in the genomes were verified by PCR experiments using a previously reported procedure (Sakuma et al., 2018 (link)). Briefly, PrimeSTAR GXL DNA Polymerase (Takara Bio, Otsu, Japan) was used for amplification. The reaction mixture, containing 60 ng genome DNA, was denatured at 98°C for 1 min and then subjected to 40 cycles, consisting of 98°C for 10 s, 61°C for 15 s, and 68°C, for 1 min. The amplified products were electrophoresed on an agarose gel and visualized with the Gel Doc EZ imager (Bio-Rad, Hercules, CA, United States). Then, 1 kb Plus DNA Ladder was used as a molecular marker (Thermo Fisher Scientific, Waltham, MA, United States).

With minimal restraints, blood samples (2-3 mL) were collected from the venous vessel of each cat. Then, these samples were stored at −20°C until DNA extraction. Subsequently, nucleic acid was extracted from blood samples (200 μL) using a DNA extraction kit (Blood Genomic DNA Extraction Mini Kit, Favorgen, Taiwan) followed by polymerase chain reaction (PCR), using the modified protocol by Godiksen et al. [17 (link)]. Briefly, the forward primer was 5′-AGCCTTCAGCAAGAAGCCA-3′, and the reverse primer was 5′-CAAACTTGACCTTGGA-GGAGC-3′. The PCR process was also conducted using a Thermocycler T-Gradient ThermoBlock (Biometra, Thailand). First, PCR steps were conducted at 95°C for 15 min, 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, and final extension at 72°C for 10 min. Then, PCR products at 242 bp on a 1.5% agarose gel electrophoresis were detected and purified using a PCR product purification kit (GEL/PCR Purification Kit, Favorgen) followed by storage at −20°C. Next, Sanger’s sequencing was performed to detect the PCR product’s nucleotide with a specific forward and reverse primer. Finally, MYBPC3 p.A31P and p.A74T polymorphisms were detected using BioEdit 7.2 (https://bioedit.software.informer.com/) and A plasmid Editor programs (jorgensen.biology.utah.edu/wayned/ape/).

The measurement of HR activity by ASHRA was described previously (17 (link)). In brief, cells grown in 3.5 cm dishes were transfected with siRNA against BRCA1 and the HA-tagged BRCA1 expression vector. On the next day, the donor vector (Addgene ID: #169798) and the expression vector for gRNA and Cas9 (Addgene ID: #169795 and #169796; 0.5 μg each) were cotransfected into the cells. After 48 hours of incubation, genomic DNA was extracted using the Blood Genomic DNA Extraction Mini Kit (Favorgen). Quantitative PCR was performed on a CFX96 Touch Real-time PCR detection system (Bio-Rad) using GoTaq qPCR master mix (Promega). Quantification of the knocked-in allele and control allele by qPCR was performed using the following primer sets: 5′-GTCCTGCTGGAGTTCGTGACCG-3′ and 5′-GTGCAATCAAAGTCCTCGGC-3′ for the knocked-in allele, and 5′-AGTTGCGTTACACCCTTTCTTG-3′ and 5′-GTGCAATCAAAGTCCTCGGC-3′ for the control allele. The relative quantity of the knocked-in allele was calculated by the ΔΔC_t method.