Generating linked transcriptional and surface marker profiles for more than 1600 single HSPCs. (A) Schematic of the sorting strategy that was used paired with index sorting data. Bone marrow cells were stained with 9 antibodies against various cell surface markers to isolate HSPCs (Lin⁻c-Kit⁺Sca1⁺ [L⁻S⁺K⁺]) and progenitors (Lin⁻c-Kit⁺Sca1⁻ [L⁻S⁻K⁺]). Almost all cells in the Flk2-CD34 gate and the CD16/32-Flk2 gate were collected for HSPCs and progenitors, respectively, within broad, all-encompassing gates. In addition, LT-HSCs (Lin^–c-Kit⁺Sca1⁺CD34^–Flk2^–) were collected separately to ensure that adequate numbers were collected. Each cell population retrospectively identified is shown in the table; colors and names remain consistent throughout the text. Letters indicate populations in the flow cytometry diagrams. (B) Unsupervised hierarchical clustering of gene expression data for all cells. Clustering was performed by using all 4773 variable genes except Ly6a/Sca-1 to avoid bias in clustering. The cells split into 4 major clusters (cluster 1, purple; cluster 2, turquoise; cluster 3, gold; cluster 4, pink). The top 10 genes enriched in each cluster are displayed in the heat map, showing gene expression on a log₂ scale from blue to orange (low to high). The clusters were also compared by cell type composition, following both broad and narrow gating strategies. Broad gating involved the classification of all cells into a cell type category, whereas narrow gating included only cells that are more likely to fit the predefined HSPC classification, gated around the greatest density of cells within the population gating strategy. Cell type is colored on the basis of the scheme used in Figure 1A. Gray cells in the narrow gating strategy represent cells unassigned to any population. FACS, fluorescence-activated cell sorting; FSC-H, forward-scattered light-height; Prog, progenitor.