title: GSM4832704: HEK293T NAC-rescued 2; Homo sapiens; OTHER
library_construction_protocol: Subcellular fractionation was performed as previously described (Arnoult et al., 2003) with some modifications. Briefly, cells were washed with PBS, harvested by scraping and centrifuged at 1000 g for 5 min. The pellet was re-suspended in buffer containing 210 mM sorbitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES and 0.1% BSA (Sigma) before grinding with a Dounce Homogenizer (Wheaton, USA) with a loose and tight pestle (100 strokes with each pestle). Cells were observed under microscope (Axiovert 40C, Zeiss) with trypan blue dye to assess cell membrane disruption followed by centrifugation at 500 g for 5 min at 4 °C. The supernatant was collected before centrifugation at 10 000 g for 30 min at 4 °C. DNA extraction (Nucleospin Tissue, Macherey-Nagel) was performed on the resulting pellet according to manufacturer instructions. mtDNA was digested using BamH1 HF (New England BioLabs) in order to linearize mtDNA genome. 400ng of DNA from each sample was barcoded and multiplexed using the Nanopore Rapid Barcoding Sequencing kit (SQK-RBK004) according to manufacturer's instructions. Sequencing was conducted with a Minion sequencer on ONT 1D flow cells (FLO-min106) with protein pore R9.4 1D chemistry for 48h.