yHSC and oHSC scRNAseq with oHSC AThi ATlo Index Sorting. Assessing the transcriptional changes associated with autophagy engagement in old hematopoietic stem cells at single cell resolution.

10X genomics single cell analysis of yHSC vs oHSC from Gfplc3/Gfplc3 mice with index sorted SMART-Seq2 sequencing to map AThi oHSC vs ATlo oHSC based on GFP-LC3 marker levels. 10X Genomics data harmonized by nearest neighbor integration and uniform manifold approximation and projection (UMAP) representation distinguished yHSC from oHSC, with the largest transcriptional differences observed in the G0/G1 cell cycle phase cluster. Within oHSCs, AThi oHSCs were almost exclusively observed in the G0/G1 cluster, whereas ATlo oHSCs were spread across the activation continuum, with cells still in the G0/G1 cluster found more proximal to S cluster cells. Overall design: For 10X genomics, two biological replicates each of yHSC and oHSC were preprared using 30,000 cells per sample. For SmartSeq2, 96 HSCs from the same samples were sorted into individual wells recording information on surface markers and GFP-LC3 level.