Fig. 4

LOY detection by digital PCR. A gDNA was extracted from male hTERT-RPTEC or female HEK293T cell lines and mixed in the indicated male: female (M:F) ratios. The mixed DNA samples were measured by multiplex dPCR using a FAM-labeled chrY target and ABY-labeled chrX reference. The measured copy ratio is the number of microwells that amplify the chrY target divided by the number of microwells that amplify the chrX reference. These values were compared to the expected copy ratio, which was estimated based on the relative ratio of M:F DNA input. For example, a 50:50 mixture of M:F DNA would yield 1 copy of chrY for every 3 copies of chrX and an expected copy ratio of 0.33. B gDNA was extracted from male kidney cortex samples and assayed by dPCR as previously described. Each sample was assayed with 2 technical replicates and 2 independent batches for a total of 4 measurements per sample. The sample labeled control is gDNA isolated from a male hTERT-RPTEC cell line. C We used dPCR to estimate the fraction of male cells with LOY using the following formula: 1 − chrY/chrX. This approach assumes the concentration of chrX is constant and that any changes in the measured copy ratio are due to changes in the concentration of chrY. We compared our dPCR and single-cell estimates to compute an r-squared value using the lm function in R