All cell lines were maintained at 37°C in a humidified incubator with 5% CO2. The human osteosarcoma thymidine kinase-deficient cell line 143B(TK−) (ATCC CRL-8303) and human embryonic kidney HEK293 cells were maintained in Dulbecco's Modified Eagle Medium GlutaMAX (DMEM; Thermo Fisher Scientific, 10569044) containing high glucose (4.5 g/L) and sodium pyruvate (110 mg/L), supplemented with 1% non-essential amino acids (NEAA) and 10% fetal bovine serum (FBS). The human lung adenocarcinoma cell line A549 and the WAl2A WAL2A lymphoblastoid49 cell line were cultured in RPMI 1640 medium with ATCC modifications (Thermo Fisher Scientific, A1049101), supplemented with 1% NEAA and 10% FBS. Corresponding mitochondrial DNA-depleted (ρ⁰ or Rho0) derivatives were maintained in the same medium supplemented with 50 μg/mL uridine (Sigma-Aldrich, U3750-100G). Adherent cell lines were passaged at 70-95% confluence by washing with PBS (Thermo Fisher Scientific, 14190250) and dissociating with 0.25% trypsin-EDTA (Thermo Fisher Scientific, 25200114). Cells used for comparative experiments were harvested at the same passage number. Single-cell RNA-seq Library Preparation For the WT and Rho0 samples, an equal mixture of four cell lines (143B, WAL2A, HEK293, A549) was processed using the Chromium Single Cell 3′ Reagent Kit v3.1 (10x Genomics). Cell barcoding, reverse transcription, cDNA amplification, and 3′ gene expression library preparation were performed according to the manufacturer's protocol. PacBio Library Preparation Genomic DNA was extracted from cell pellets and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q33231). Full-length mitochondrial genomes were amplified by long-range PCR using using PrimeSTAR GXL DNA Premix (Takara, R051B) with custom primers and reaction conditions as previously described50. PCR products were purified with SMRTbell Cleanup Beads (PacBio, 102-158-300), and assessed for fragment size distribution using the Femto Pulse system (Agilent, FP-1002-0275). PacBio libraries were prepared using the SMRTbell Prep Kit 3.0 (PacBio, 102-182-700) according to their standard protocol. Following adapter ligation and nuclease treatment, libraries were size-selected using AMPure PB beads (PacBio, 100-265-900). Libraries were annealed to sequencing primers using the Sequel II Binding Kit 3.2 (PacBio, 102-333-300), bound to Sequel II Polymerase 2.2, loaded onto SMRT Cell 8M (PacBio, 102-281-700).