title: Optimizing single cell mitochondria sequencing in a microfluidic platform and identifying mtDNA variations in aging mice tissues
design: First of all, we proposed a gentle lysis of the cell using 0.1% (w/v) NP40 before ordinary single cell processing in the DNBelab C4 single cell ATAC protocol [ref], which was our first possible protocol. Gentle lysis of the cell would ideally not break down the cell membrane thus mitochondria should be remained in cell and subsequent process would isolate cells and DNA in mitochondria of each cell can be transposed together with the genomic DNA and finally subjected to sequencing. Secondly, referring to the previous single cell mtDNA method, fixation of the mitochondria using formaldehyde can reduce the cross contamination between mitochondria from different cells. Thus, we proposed the second and third protocols to use formaldehyde to cross-link the mitochondria in cells before the gentle cell lysis and subsequent ordinary single cell ATAC protocol. We used a formaldehyde concentration of 1% in the second protocol and a formaldehyde concentration of 0.1% in the third protocol. In addition to these three possible protocols, we also used the ordinary single cell ATAC protocol in which Tween-20, digitonin and NP40 are used for cell lysis as control.