Optimizing single cell mitochondria sequencing in a microfluidic platform and identifying mtDNA variations in aging mice tissues

Microfluidic systems have been developed to reveal transcriptome and open chromatin in single cell level. However, they were not commonly used to massively study single cell mitochondrial DNA (mtDNA) variations, which represent the mtDNA heteroplasmy, and might be functionally important. Here, we applied the microfluidic single cell sequencing, DNBelab C4 to handle the cells, and then optimized the protocol to sequence mtDNA in single cells. We first established a workflow of using whole cell instead of nucleus for mild lysis to release mtDNA, and fixation of the mtDNA in each cell, followed by single cell barcoding and finally sequencing. We then assessed and obtained an optimized workflow with mtDNA proportion reaching up to 67.793% and the collision rate as low as 1.89%. Next, we applied our workflow to identify possible single cell mtDNA variations of aged mice tissues. The VAF was below 1% and more than 70% of cells carried mtDNA variations in aged mice spleens and bone marrows, and mtDNA variations varied in different cells. 76.9% and 58.8% of the mtDNA variations were in protein-coding genes, of which 66.7% and 47.1% were nonsynonymous, respectively. Changes in gene expressions and signaling pathways were uncovered between the cells with and without mtDNA variations. Interestingly, m.11185G>A encoding NADH-ubiquinone oxidoreductase chain 4 was identified in both aged spleens and bone marrow, which was associated with Leber hereditary optic neuropathy (LHON). Our study would benefit future single cell studies by providing an alternative solution for single cell mtDNA sequencing.