Maxwell 16 system dna purification kit

Genotyping and copy number analysis of SOD1^G93A mice was performed based on the protocol described in Mead et al.⁴⁹ (link) Any changes made are detailed below.
Mouse genotyping was performed on genomic DNA extracted from tail tissue using the Maxwell 16 system DNA purification kit (Promega, WI, USA). Genotyping PCRs were performed on genomic DNA in a 25-mL volume with ABgene ReddyMix (ABgene, Epsom, UK), 150 nM each of hSOD1 primers (forward, 59-CATCAGCCCTAATCCATCTGA-39; reverse, 5′-CGCGACTAACAATCAAAGTGA-3′) and control interleukin-2 receptor (IL-2R) primers (forward, 5′-CTAGGCCACAGAATTGAAAGATCT-3′; reverse, 5′-GTAGGTGGAAATTCTAGCATCATC-3′). Following PCR and agarose gel electrophoresis (3% agarose gel), IL-2R PCR products were visualized at 324 bp and hSOD1, if present, at 236 bp. Mice positive for transgene expression were then subjected to copy number analysis by qPCR using 30 ng cDNA, 2× SYBR Green PCR Master Mix (Agilent Technologies, CA, USA), and 150 nM primers used for genotyping, in a total volume of 20 mL. Following an initial denaturation at 95°C for 10 min, DNA was amplified by 40 cycles of 95°C for 15 s and 60°C for 1 min, on an MX3000P Real-Time PCR System (Stratagene). hSOD1/IL-2R ΔCt values from samples were compared to a reference DNA sample from an SOD1^G93A control mouse displaying a phenotype consistent with one carrying the full 25 copies of the transgene.

After extraction of genomic tumoral and non-tumoral DNA from frozen samples using a Promega Maxwell® Instrument with the Maxwell® 16 System DNA Purification Kit. DNA was quantitated using Hoechst dyes and a microplate reader. The detailed process for enrichment of individual genomic DNA (gDNA) sample libraries is shown in Supplementary materials (using SureSelect XT Target Enrichment System). After qualifying libraries of tumor and non-tumor tissues, they were sequenced by SureSelectXT Human All Exon V6 (target size 60 Mb, Agilent, Santa Clara, CA) and paired-end sequencing was carried out on a Illumina HiSeq X Ten, PE150 with 2 × 150 base pairs (bp) read length. Raw data and base calls were processed using standard Illumina Miseq Reporter software (version 2.5.1). The downstream data analysis was done on the resulting Illumina FASTQ files. The reads were aligned on human hg19 genome reference utilizing BWA version 0.7.5a and bam files were generated using samtools v1.3.