Magmax dna multi sample ultra 2.0 kit

High-throughput isolation of DNA was performed using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on a KingFisher Flex robotic DNA isolation system (Thermofisher, Waltham, MA) according to manufacturer protocol. Briefly, 20–40 mg of fresh frozen brain tissue was placed into a deep-well plate and treated with 480 ul of Proteinase K mix (Proteinase K, Phosphate Buffered Saline [pH 7.4], Binding Enhancer) and incubated overnight at 65 °C at 800 rpm on a shaking plate. Genomic DNA was isolated and purified using magnetic particles. DNA quality control was performed using a nanodrop spectrophotometer (concentration > 50 ng/ul, 260/280 ratio 1.7–2.2). Genotyping was performed using single nucleotide polymorphism (SNP) microarrays (Infinium Global Screening Array v2.4. or the Infinium OmniExpress-24, Illumina, San Diego CA). Raw genotype files were converted to PLINK-compatible files using GenomeStudio software (Illumina, San Diego CA). MAPT haplotype was determined using the rs8070723 H2 tagging SNP and APOE genotype was determined using the rs429358 rs7412 tagging SNPs. For analyses, the APOE status was collapsed into a binary variable of the presence or absence of APOE ε4.

HEK293T cells were nucleofected with precomplexed RNP consisting of either 12 pmol Cas9 and 60 pmol sgRNA, 104 pmol MG3-6 and 120 pmol sgRNA, or 120 pmol MG29-1 and 120 pmol sgRNA, using the Lonza 4D electroporation system. In parallel, cells were cotransfected with 50 pmol annealed dsODN. After 72 h, cells were trypsinized and gDNA extracted using the Kingfisher MagMAX DNA Multi-Sample Ultra 2.0 Kit (Catalog No. A36570). Four hundred nanograms of high molecular weight gDNA was fragmented, end-repaired, and ligated using the NEB FS DNA Library Prep Kit (Catalog No. E7805). Fragments between 350 and 600 bp in length were amplified to enrich for dsODN-proximal regions using dsODN-specific primers in both the positive and negative orientations. Resulting libraries were amplified for next generation sequencing (NGS) on Illumina Miseq.

High-throughput isolation of DNA was performed using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on a KingFisher Flex robotic DNA isolation system (Thermofisher, Waltham, MA) according to the manufacturer's protocol. Briefly, 20–40 mg of fresh-frozen brain tissue was placed into a deep-well plate and treated with 480 μL of Proteinase K mix (Proteinase K, Phosphate Buffered Saline [pH 7.4], Binding Enhancer) and incubated overnight at 65 °C at 800 rpm on a shaking plate. Genomic DNA was isolated and purified using magnetic particles. DNA quality control was performed using a nanodrop spectrophotometer (concentration > 50 ng/μL, 260/280 ratio 1.7–2.2). Genotyping was performed using single-nucleotide polymorphism (SNP) microarrays (Infinium Global Screening Array v2.4. or the Infinium OmniExpress-24, Illumina, San Diego CA). Raw genotype files were converted to PLINK-compatible files using GenomeStudio software (Illumina, San Diego CA). MAPT haplotype was determined using the rs8070723 H2 tagging SNP and APOE genotype was determined using the rs429358 rs7412 tagging SNPs. For analyses, the APOE status was collapsed into a binary variable of the presence or absence of APOE ε4.