Mapping of mtDNA and nDNA 5'-ends and ribonucleotides

The 5′-ends in mtDNA and nDNA from mouse liver were mapped using 5′-End-seq by treating 1 μg of total DNA with 0.3 M KCl for 2 h at 55 °C. Ribonucleotides were mapped by HydEn-seq (30 (link)) by hydrolyzing 1 μg of total DNA with 0.3 M KOH for 2 h at 55 °C. Afterward, ethanol-precipitated DNA fragments were treated for 3 min at 85 °C, phosphorylated with 10 U of phosphatase-free T4 polynucleotide kinase (New England BioLabs) for 30 min at 37 °C, heat-inactivated for 20 min at 65 °C, and purified with HighPrep PCR beads (MagBio). Phosphorylated products were treated for 3 min at 85 °C, ligated to oligo ARC140 (30 (link)) overnight at room temperature with 10 U of T4 RNA ligase, 25% polyethylene glycol (PEG) 8000, and 1 mM CoCl₃(NH₃)₆, and purified with HighPrep PCR beads (Mag-Bio). Ligated products were incubated for 3 min at 85 °C. The ARC76 ± ARC77 adaptor was annealed to the fragments for 5 min at room temperature. The second strand was synthesized with 4 U of T7 DNA polymerase (New England BioLabs) and purified with HighPrep PCR beads (MagBio). Libraries were PCR-amplified with KAPA HiFi Hotstart ReadyMix (KAPA Biosystems), purified, quantified with a Qubit fluorometric instrument (Thermo Fisher Scientific), and 75-base paired-end sequenced on an Illumina NextSeq500 instrument to locate the 5′-ends.