Apexhf hs dna polymerase

Full-length FTH cDNA was ordered from Sino Biological (Beijing, China), amplified by ApexHF HS DNA Polymerase (Accurate Biotechnology, Hunan), subcloned into pLVX-IRES-Neo lentivirus vector by Seamless Cloning kit and verified by sequencing. The recombinant lentiviral plasmids were co-transfected with pMD2.G, pSPAX2 into 293 T cells to produce recombinant lentiviral. For transfection, HCCLM3 and MHCC97H cells were plated in a 24-well plate, and co-incubated with the corresponding lentivirus for 8 h. Then the stable cell lines were screened by G418 (1000 μg/mL) for 7 days.

The CRISPR array and tracrRNA region of Lactobacillus rhamnosus GG was PCR amplified by ApexHF HS DNA Polymerase (Accurate Biology, China) and TA-cloned into pEASY-T1 cloning kit (TransGen, China), following the manufacturer’s instruction. The colonies confirmed by Sanger sequencing were cultured in the flask for 16 h. The cultured bacteria were collected by centrifuge and remove the supernatant. After fast freezing by liquid nitrogen, the samples were sent to Novogene (China, Tianjin) for RNA sequencing using the Novaseq6000 platform. The histogram was exported by the Integrative Genomics Viewer (IGV)^{88 (link)} software.

The mutation frequency in protoplasts was analyzed by amplicon deep-sequencing^{61 (link)}. The amplicons of the editing regions were amplified by ApexHF HS DNA Polymerase (Accurate Biology, China), and the barcodes were added through PCR primers. Amplicons were sent to Novogene (China, Tianjin) for deep-sequencing by the Novaseq6000 platform which produced 150 bp paired-end reads. The editing frequency and profile were analyzed by CRISPRMatch software^{89 (link)}. The mutation frequencies in wheat and larix were normalized to the protoplast transformation efficiencies in both species. For stable rice T₀ lines, the editing outcomes were identified by single-strand conformation polymorphism assay^{82 (link)} and direct PCR product Sanger sequencing provided by Sangon Biotech (Shanghai, China) and analyzed by CRISPR-GE DSDecodeM software^{59 (link)}.