Hindiii digest of lambda dna

For mtDNA analyses, approximately 10 mLs were drawn into PAXgene Blood DNA tubes (Qiagen, 761115, containing 2mLs of a proprietary additive which prevents coagulation of the blood and preserves genomic DNA). PAXgene tube samples for mtDNA CN and damage assays were stored in a –20°C freezer for 24 hours, transferred to a -80°C ultra-freezer, shipped to Duke University on dry ice, and stored at -80°C until processing for DNA isolation. The frozen whole blood samples were thawed in a 37°C water bath for 15 minutes and then immediately processed. PAXgene Blood DNA kits (Qiagen, 761133) were used according to the manufacturer’s instructions to extract high molecular weight DNA. DNA yield and purity were initially analyzed using a NanoDrop ND-1000 (ThermoFisher), then further quantified using PicoGreen (ThermoFisher P7589) with a standard curve of a HindIII digest of lambda DNA (Invitrogen 15612–013) as described [55 (link), 56 (link)]. This results in isolation of very high-quality, high-molecular weight total genomic DNA required for our DNA damage assay [57 (link)]. This isolated DNA was used for CN and damage assays described below.

For mtDNA analyses, approximately 10 mLs were drawn into PAXgene Blood DNA tubes (Qiagen, 761115, containing 2mLs of a proprietary additive which prevents coagulation of the blood and preserves genomic DNA). PAXgene tube samples for mtDNA CN and damage assays were stored in a –20°C freezer for 24 hours, transferred to a -80°C ultra-freezer, shipped to Duke University on dry ice, and stored at -80°C until processing for DNA isolation. The frozen whole blood samples were thawed in a 37°C water bath for 15 minutes and then immediately processed. PAXgene Blood DNA kits (Qiagen, 761133) were used according to the manufacturer’s instructions to extract high molecular weight DNA. DNA yield and purity were initially analyzed using a NanoDrop ND-1000 (ThermoFisher), then further quantified using PicoGreen (ThermoFisher P7589) with a standard curve of a HindIII digest of lambda DNA (Invitrogen 15612–013) as described [55 (link), 56 (link)]. This results in isolation of very high-quality, high-molecular weight total genomic DNA required for our DNA damage assay [57 (link)]. This isolated DNA was used for CN and damage assays described below.