Ultra pure agarose 1

Full-length mtDNA amplification was carried out for short-read and long-read sequencing, and as a part of quality control using Platinum SuperFi II DNA Polymerase (Invitrogen). A forward primer Mt2120F (5′-GGA CAC TAG GAA AAA ACC TTG TAG AGA GAG -3′) and a reverse primer Mt2119R (5′-AAA GAG CTG TTC CTC TTT GGA CTA ACA -3′) were used. All lrPCRs (50 μl) were prepared as follows: 1× SuperFi II buffer, 0.4 mM each of dNTPs, 0.2 μM of each forward and reverse primers, 1× of SuperFi II DNA Polymerase, 0.5 mM MgCl2, and 10-100 ng of individual DNA sample. Cycling conditions were: 94°C for 1 min; 30 cycles of 98°C for 10 s, 68°C for 16 min; 72°C for 10 min; hold at 4°C. To visualize amplicons, Ultra Pure Agarose 1% (Invitrogen) gel was prepared in 1× TAE buffer (BIO-RAD). DNA samples were loaded together with Quick-Load 1 kb Extend DNA Ladder (NEB) to aid size determination.
Fragments were separated running a gel at 60 V for 2.5-3 h. Amplicons were purified with AMPure XP beads (Agencourt, Beckman Coulter) and eluted in 30 μl nuclease-free water. Concentration was measured using a Qubit fluorometer with the dsDNA Broad Range Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. incubating at 37ºC for 20 min followed by Quick CIP inactivation at 80ºC for 3 min. For the following Cas9-mtDNA-enrichment procedure each DNA sample was split into aliquots.