Single-cell gene expression analysis of mouse bone marrow cells

Cells were sorted from mouse bone marrow as described above and analyzed for gene expression as performed previously (Reynaud et al., 2011 (link); Pietras et al., 2015 (link); Pietras et al., 2016 (link); Hernandez et al., 2020 (link); Rabe et al., 2020 (link); Chavez et al., 2021 (link); Ahmed et al., 2022 (link); Chavez et al., 2022 (link)). Cells were sorted at 100 cells per well in 5 μL CellsDirect reaction buffer (Invitrogen). RNA was then reverse transcribed and preamplified with a panel of 96 DeltaGene Assay primer sets (Fluidigm) for 20 rounds with Superscript III (Invitrogen) and subsequently treated with Exonuclease I (New England Biolabs) to remove non-target genetic material. cDNA was then diluted in DNA suspension buffer and loaded onto Fluidigm 96.96 Dynamic Array IFCs along with the DeltaGene Assay primers and run on a Biomark HD (Fluidigm) using SsoFast Sybr Green (Bio-Rad) as a detector. Data were analyzed using the ΔΔCT method and normalized to Gusb expression. Hierarchical clustering and PCA analyses were performed using ClustVis. As Gusb was used to normalize data, it was not included in clustering and PCA analysis. A complete list of all Fluidigm primer sequences can be found in Supplementary Table S4. Hoxa2 and Ebf1 were excluded from all analyses due to poor primer performance in our studies.