Single-cell RNA-sequencing of differentiation myeloid cells

During granulocyte-macrophage colony-stmulating factor (GM-CSF) driven ex vivo myeloid differentiation of mouse hematopoietic stem/progenitor cells defined as Lineage negative cKit positive Sca-1 postive (LSK) cells, LSK cells after Cxxc5 knockdown makes more granulocytic cells than monocytic cells. To study this effect of Cxxc5 knockdown during myeloid cell differentiation, we performed a single-cell RNA sequencing of differentiating myeloid cells and analyzed the transcriptome of these cells. Stem/progenitor cells enriched bone marrow cells from five C57BL/6 mice were infected with retrovirus to knockdown Cxxc5. Retrovirus that does not target any mouse genes was used as a control. Infected LSK cells (GFP positive LSK) were flow-sorted and cultured in a differentiation media containing cytokines GM-CSF and SCF. Cells were collected three days post growth in the myeloid differenation media, pooled (5 control samples pooled as one control and 5 knock-down samples pooled as one knock-down sample) and used for single-cell RNA sequencing with 10X Genomics.