Single cell RNA seq of CD34+ cells isolated from fetal bone marrow of GD135 fetuses born to dams on Chow or WSD
Freshly thawed CD34+ HSPCs were enriched for live cells by FACS using SYTOX Blue dead cell stain (ThermoFisher) on BD FACS Aria Fusion. Cells were sorted into RPMI containing 30% FBS, 5% glutamine, and antibiotics. Cells were then counted in triplicates on a TC20 Automated Cell Counter (BioRad), washed and resuspended in 1X PBS with 0.04% BSA in a final concentration of 1200 cells/uL. Single cell suspensions were then immediately loaded on the 10X Genomics Chromium Controller with a loading target of 17,600 cells. Libraries were generated using the 3 V3 chemistry per manufacturers instructions (10X Genomics, Pleasanton CA) and sequenced on Illumina NovaSeq with a sequencing target of 30,000 reads per cell.