Illumina HiSeq 3000 sequencing; DNA-seq of mitochondrial DNA from mouse heart that is both heart SOD2 knockout and deficient for mitochondrial DNA repair
DNA-seq of mitochondrial DNA from mouse heart that is both heart SOD2 knockout and deficient for mitochondrial DNA repair
Fresh tissue was minced to small pieces (~100 mg) washed in PBS and mitochondrial DNA was purified as described in the nucleic acid extraction protocol. First, fresh tissue was minced to small pieces (~100 mg heart, 400-500 mg liver) washed in PBS and homogenized into 10 ml of mitobuffer (320 mM sucrose, 20 ml Tris-HCl, 1 mM EGTA, 0.2% BSA, pH 7.2) with a glass homogenizer. Heart tissue was first homogenized with loose Teflon pestle by hand with 10 strokes. Then, both liver and heart were homogenized with a tight Teflon pestle with 5 (liver) or 10 strokes (heart) at 200 rpm. Next, homogenates were spun 10 min at 800 g at 4°C to pellet the cell debris, followed by a 10 min spin at 8 500 g 4°C to pellet the mitochondria. The mitochondria pellet was resuspended in 600 µl of Mito-DNase buffer (300 mM sucrose, 10 mM MgCl2, 20 mM Tris HCl, pH 7.5, 0.15% BSA, 0.03 mg/ml DNase I type IV, 170 ng/µl RNase A) and the liver mitochondria were further divided into 600 µl aliquots each containing ~100 mg of starting material. The suspensions were incubated at 37°C for 1 hr to digest the nuclear DNA and RNA. Then, mitochondria were repelleted at 13 000 g, for 15 min at 4°C and washed twice with 500 µl of mitobuffer (320 mM sucrose, 20 ml Tris-HCl, 1 mM EGTA, 0.2% BSA, pH 7.2). Next, the samples were frozen in N2(l) and stored in -80°C. Later the same day, the mitochondrial pellets were resuspended into 400 µl lysis buffer (20 mM Tris HCl, 150 mM NaCl, 20 mM EDTA, 1% SDS, pH 8.75, 0.2 mg/ml Proteinase K, 0.2 mg/ml RNase A) and incubated at 56°C overnight. Then, the samples were cooled down to RT and DNA was extracted with chloroform (100 µl of 6M K-acetate, 500 µl chloroform:isoamylalcohol (24:1, Amresco)). 100 µg/200 µg of RNase A was added to each aqueous phase fractions (heart and liver, respectively) and samples were incubated at 37°C for 45 min to digest the remaining RNA. Thereafter, the samples were ethanol precipitated with 15 µg of glycogen (Ambion). The purified DNA pellet was then resuspended to 20-30 µl of 5 mM Tris buffer pH 8.5 (Macherey-Nagel). Prior to sending the samples to Illumina sequencing (Max Planck Genome Center Cologne), the purity of the extracted mtDNA was verified using mtDNA and nuclear DNA specific primers for PCR amplification. The yield from each 100 mg fractions were ~250 ng for heart and ~ 350 ng for liver as quantified with a fluorometric method (Qubit, ThermoFisher). The protocol was modified from a previously described protocol (Kennedy et al 2013 Plos Genetics, PMID 24086148). For details see (dx.doi.org/10.17504/protocols.io.mycc7sw)  Isolated mtDNA from the hearts were sequenced in two separate sets each including controls. First, the quality of the samples was verified with Genomic DNA analysis ScreenTape (Agilent) followed by DNA library preparation. The DNA was fragmented with Covaris to 400 bp (50 ng, 50 µl, 5% duty cycle, intensity 5, 200 cycles per burst, treatment time 55 s).  Samples with 2269 in their name were fragmented to 350 bp: 50 µl vol, 5 % duty cycle, intensity 5, 200 cycles per burst and  treatment time 53 s. Following DNA fragmentation, the DNA library was prepared with NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs).
Experimental Factor: phenotype
wild type phenotype