mtATAC_C57BL6_mtPWD_hepatocytes_mixing_1
C57BL6/J and mtPWD hepatocytes were sorted into 384-well plates containing 0.45ul of TD buffer. Each well received different number of C57BL6 and PWD hepatocytes, but around 10 cells in total. The uncertain cell number is due to inefficient FACS sorting. Sample 1_S1_L001 received only mtPWD hepatocytes, Samples 2_S2_L001 through 11_S1_L001 received increasing number of C57BL6 hepatocytes and decreasing number of mtPWD hepatocytes with sample 11_S11_L001 receiving only C57BL6 hepatocytes. Next, 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 11 wells.