Exonuclease 1

High-throughput gene-expression analysis with EvaGreen dye on a BioMark HD real-time PCR system was used to measure gene expression according to manufacturer’s recommendations (Fluidigm, San Francisco, CA, USA). All reagents were purchased from Fluidigm unless otherwise indicated. Briefly, cDNA was synthesized from 2 µg of total RNA using Superscript II reverse transcriptase and oligo-dT (Invitrogen) with specific target amplification and exonuclease I (New England Biolabs) treatment. qPCR reactions were performed using a 96 × 96 dynamic array and integrated fluidic circuit. Each sample inlet was loaded with 2.5 µL of 2 × SsoFast EvaGreen supermix with low ROX (Biorad), 0.25 µL of 20×DNA-binding dye sample loading reagent, and 2.25 µL of specific target amplification and exonuclease-I-treated sample. Assays were performed for mitochondrial-coded complex I subunits ND1, ND2, ND3, and ND4, complex III subunit CYB, complex IV subunit COX1, ATP synthase subunit ATP6, and nuclear-encoded complex I subunits NDUFA3, NDUFA11, NDUFA13, NDUFB8, NDUFS8, and NDUFV1, complex IV subunit COX7A2, ATP synthase subunits ATP5G1 and ATP5L, and master regulator PGC-1α. All measurements were performed in duplicate. Primer sequence information is available in Supplementary Table 4. Reference genes ACTB, RPL32, and RPS11 were used to normalize expression values.