title: mtATAC_24mo_WT_mouse_1_hepatocytes
design: Frozen 384-well plates with single hepatocytes in 0.45ul of TD buffer were thawed on ice. Hepatocytes were lysed and DNA was tagmented in a single step. To this end, 0.45 ul of lysis solution (1% n-Dodecyl β-D-maltoside [final concentration 0.5%], 90 mM NaCl [final concentration 45 mM], 10mM TrisHCl pH 8.0, 5 mM MgCl2, 10% dimethylformamide) supplemented with Tn5 (Illumina, 20034197; 1.5 ul of enzyme for 150 ul of lysis solution) was added to each well of a plate with help of Echo 525. Then, plates were sealed with foil (Bio-Rad, MSB1001), briefly centrifuged, and incubated at 37°C for 30 min. After lysis and tagmentation, Tn5 was stripped off DNA. To this end, 0.1 ul of 2% SDS was added to each well of a plate (final concentration 0.2%) using Echo 525, plates were sealed with a foil, briefly centrifuged, and incubated at 65°C for 15 min. Next, mtATAC libraries were constructed by PCR amplification of DNA fragments created by Tn5 with unique dual index primers for each well of a plate. To this end, PCR master mix (NEB, M0544S), tween-20 (final concentration 0.34%) and unique dual index primers (final concentration 500 nM; sequences are provided in Table S1; IDT) were added to each well of the plate using Echo 525 (final volume 3 ul), plate was sealed with a foil, briefly centrifuged and thermocycled as follows: incubation at 72°C for 5 min to fill the gaps; initial denaturation at 98°C for 30 s; 16 cycles of denaturation at 98°C 10 sec and combined annealing-extension at 65°C 75 sec; final extension at 65°C 5 min. Incubation and PCR were performed in a standard thermocycler (Bio-Rad, C1000 Touch or S1000). Uniquely labeled libraries from one or several plates were pooled together at equal volumes and cleaned up using home-made SPRI beads (Rohland and Reich, 2012) twice. The first cleanup was one-sided with 1.2 beads to library volume ratio. The second cleanup was two-sided with 0.5 ratio followed by 1.2 ratio. Cleaned up libraries were eluted in 20 ul of TE buffer. To quality control and quantify libraries 1 ul of cleaned mtATAC library was run on Bioanalyzer (Agilent). mtATAC libraries were sequenced on MiSeq (Illumina) using MiSeq Reagent Kit v2, 300-cycles (Illumina, MS-102-2002) as 151x12x12x151. Deposited bam file contains data from all sequenced cells of the sample that had at least 10,000 reads mapping to mtDNA (n = 360 cells). Reads from each cell have unique sample name, e.g. 1_S1_L001.