AThi oHSC ATlo oHSC WES - Identifying clonal differences or differences in mutation burden between autophagy engaging and non-engaging hematopoietic stem cells during mouse aging.

We performed paired WES from DNA isolated from AThi and ATlo hematopoietic stem cells (FACS sorted on the basis of GFP marker intensity, 33% GFPlo as ATHi, 33% GFPhi as ATlo) using tail DNA as an internal control to identify somatic mutations. Few mutations were called at a variant allele frequency of = 5% in this bulk sequencing approach, in line with recent results obtained with more sensitive methods. We also observed a high degree of shared mutations between AThi and ATlo oHSCs within each biological replicate, and no conserved mutations across biological replicates. The majority of called mutations were intronic or synonymous single nucleotide variants with limited expected consequences for their encoded proteins. Taken together, these results indicate that differing autophagy levels in oHSCs are not explained by clonal divergence or differential mutagenic burden, nor do they point to mutagenic burden as a driver of oHSC dysfunction in 24-month-old mice. They also do not provide evidence for clonal evolution between AThi and ATlo oHSC subsets. Overall design: A total of 6 paired AThi/ATlo oHSC biological replicates were sequenced alongside tail DNA from each old Gfp-Lc3 mouse to establish baseline germline mutations.