Snapgene viewer program

To confirm the VPS13B genotype, all samples were subjected to direct DNA sequencing. The VPS13B gene segment encompassing the mutation site was amplified using the F1 and R1 primers. The PCR reaction mixture and amplification protocol were similar to those used for the MAS-PCR assay, with the exception that annealing at 58°C for 2 min was used. Amplified products were then purified using a NucleoSpin^® Gel and PCR Clean-up Purification Kit (Macherey-Nagel, Germany) according to the manufacturer’s protocol. DNA was then sequenced by Macrogen Inc. (Korea) using the F1 primer. Sequence analysis was performed using the SnapGene^® Viewer program (SnapGene^® Software, GSL Biotech LLC, USA). Finally, sequence similarity comparisons were determined using the NCBI database (https://blast.ncbi.nlm.nih.gov/blast.cgi).

A total of 224 samples were submitted for direct DNA sequencing to confirm NHEJ1 genotypes. The wild-type primer set (F3 and R3) and the mutant primer set (F7 and R7) were used to amplify a fragment harboring the wild-type and the mutated allele, respectively. The PCR amplification reactions were conducted as described above. The amplified PCR products were purified with a NucleoSpin^® Gel and PCR Clean-up purification kit (Macherey-Nagel, Germany) according to the manufacturer’s instruction and then sequenced by Macrogen Inc. (Korea) using the forward primers (F3 and F7). Sequence data were analyzed using the SnapGene^® Viewer program (SnapGene^® Software, GSL Biotech LLC, USA). The sequence similarity was compared with the National Center for Biotechnology Information database (NCBI; https://blast.ncbi.nlm.nih.gov/blast.cgi).

The genomic DNA of cell cultures was isolated from cell culture samples containing 5 million cells per sample using the ExtractDNA Blood & Cells kit (Evrogen, Moscow, Russia) in accordance with the protocol provided by the manufacturer.
Gene fragments containing regions of interest for IDH1 were amplified using DreamTaq Green PCR Master Mix (2X) (Thermo Fisher, Waltham, MA, USA).
The PCR mixture was purified using the Cleanup Mini kit (Evrogen, Moscow, Russia).
For Sanger sequencing, we used 10 ng of purified DNA by BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher, Waltham, MA, USA)
The sequencing reaction was purified using the BigDye XTerminatorTM Purification Kit (Thermo Fisher, Waltham, MA, USA) and sequenced on a 3500 genetic analyzer (Thermo Fisher, Waltham, MA, USA).
The raw data results were analyzed using the SnapGene Viewer program (GSL Biotech, San Diego, CA, USA).