design: Cell cultures were maintained at 37°C in a 5% CO2 humidified atmosphere. CD34+ hematopoietic stem cells were acquired from the Fred Hutch Hematology core. Frozen CD34+ cells were thawed in serum-free RPMI plus 10mM HEPES, then cultured at 1 x 10^6 cells/ml of STIF medium [Stemspan II medium supplemented with 100 ng/ml stem cell factor, 100 ng/ml thrombopoietin, 100 ng/ml Flt3 ligand (all from Stemcell Technologies), and 100 ng/ml insulin-like growth factor binding protein 2 (R&D Systems)]. Cells were expanded at two and four days post-thaw. Seven days post-thaw, cells were processed for single-cell submission or transferred from STIF into macrophage-stimulating medium (MSM) [SFEM supplemented with 1x Myeloid Expansion Supplement II (Cat# 02694, StemSpanTechnologies) and 20µg/mL IL-6 (R&D Technologies)] for in vitro differentiated macrophages. Differentiating cells were cultured for an additional 7 days in MSM. HSCs were expanded in MSM on day 7 by seeding cells at 250k/well in 24-well plates in 500ul MSM. On day 10, cells were divided four times and maintained in 500µl MSM. On day 14, cells were collected for single-cell analysis. Cells were washed in 1 x PBS-/- supplemented with 0.04% BSA and sorted using the BigFoot cell sorter (Thermo Fisher) on days 7 or 14. For in vitro-differentiated adherent cells, cells were lifted using enzyme-free cell dissociation buffer (ThermoFisher) before washing and sorting. Nuclei were isolated following the 'Low Cell Input Nuclei Isolation' sub-protocol and immediately processed using the Chromium Next GEM Single Cell Multiome + Gene Expression kit. Single Cell Multiome ATAC and Gene Expression libraries were prepared according to the 10X Chromium Next GEM Single Cell Multiome ATAC + Gene Expression user guide for standard Illumina paired-end sequencing.