Rare non-canonical megakaryocyte progenitors increase in number and gain enhanced function with age

Rare non-canonical megakaryocyte progenitors increase in number and gain enhanced function with age Overall design: CITEseq via the 10X Genomics platform was performed simultaneously with our previously published scRNAseq data of young and aged FlkSwitch mice. Briefly, cKit-enriched BM was sorted into CD150+ and CD150- fractions and incubated with CITEseq antibodies (BioLegend) per manufacturer's instructions. All other steps as described in Poscablo et al., 2024. Chromium Next GEM Single Cell 3' v3.1 (Dual Index) reagents and protocols utilized as specified. Sequencing depth of 5,000 read pairs per cell was performed. Cells expressing fewer than 200 genes were excluded from the analysis. Cells were further filtered based on surface protein marker expression; specifically, double-negative cells for CD117 ADT and Kit RNA were excluded, removing 13 cells from the dataset. To normalize the CITE-seq data, we implemented a centered log-ratio (CLR) transformation for each cell. For each cell's antibody-derived tag (ADT) count vector, we first computed the natural logarithm of the counts, offset by one, for all non-zero elements. The sum of these log-transformed values was then divided by the total number of features, and the exponential of this mean was used to normalize the original counts. Finally, a log transformation was applied to the resulting normalized values to stabilize variance across features.