Single-cell RNA sequencing of myeloid cells, hematopoietic stem and progenitor cells, and mesenchymal stromal cells isolated from bone marrow of C57BL/6J WT and IL-1rn-KO mice

Interleukin-1β (IL-1β) drives hematopoietic stem cell (HSC) differentiation into the myeloid lineage, and enhanced IL-1β signaling plays a key role in hematological malignancies. However, little is known on the role of its endogenous regulatory cytokine, IL-1 receptor antagonist (IL-1rn), on both healthy and malignant hematopoiesis. Here, characterize transcriptomic changes at the single cell level in myeloid cells, hematopoietic stem and progenitor cells, and CD63+ mesenchymal stromal cells between C57BL/6J and IL-1rn-KO mice. We profiled bone marrow (BM) hematopoietic and non-hematopoietic cells by scRNA-seq from females C57BL6/J WT (n = 1) and IL-1RN KO (n = 1) aged 13-15 weeks. We sorted myeloid cells as CD11b+, hematopoietic stem and progenitor cells (HSPC) as Lineage- Sca-1+ cKit+ (LSK) and mesenchymal stromal cells (MSC) as CD45- CD31- Ter119- CD63+ by FACS. Sorted CD63+ MSCs were pooled from two animals to reach the desired number of cells for scRNA-seq, after separation of one femur and one tibia from one of the mice dedicated to CD11b+ and LSK cell sorting. Cells were counted and their viability checked using the Countess III cell counter (Thermofisher). Single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). Each cell suspension was loaded into one port of a Chromium Next GEM Chip G (10x Genomics) with a target output of 4000-10000 cells.