Enrichment by organelle isolation and nuclear DNA depletion: ‘mitoenrichment’
The mitochondria isolation procedure is based on sequential precipitation. We used the Mitochondria Isolation kit for cultured cells (ThermoFisher) and followed protocol B (details in S1). Subsequently, we added 2 ml of mitochondria lysis buffer (Isokallio & Stewart, 2018) and 100 µl of proteinase k to the crude mitochondria pellet and incubated up to 2 h after which the phenol chloroform DNA extraction was conducted. In order to maximize the amount of pure mtDNA, nuclear DNA (nDNA) removal was performed in a 40 µl reaction with 30 µl of DNA at ideally >60 ng µl-1, 1×NEBuffer4, 1mM ATP and 10 U of Exonuclease V (RecBCD) (New England Biolabs Inc.). The digestion was performed at 37°C for 2 h followed by heat-inactivation at 70°C for 30 min (Dhorne-Pollet et al., 2020). The resulting DNA was cleaned and concentrated using 0.7× Ampure XP (Agencourt) (with two washes with 70% EtOH) and eluted in 10 µl of TlowE. Nanopore library preparation was conducted with the rapid sequencing kit (SQK-RAD004, ONT), which uses a transposase that cleaves the DNA randomly linearizing the mitochondrial DNA while adding the necessary adapters for the motor proteins that serve as sequencing adapters using up to 400 ng oftemplate following manufacturer’s protocol (Fig 1, detailed protocol in S1). Running times varied as in Table 3.