Heterogeneity of neutrophils in tissues

To identify transcriptional diversity of neutrophils in steady state we performed single cell sequencing analysis on 13033 individual neutrophils across 6 different body compartments (bone marrow, blood, spleen, lung, liver and intestine). Dimensional reduction analysis identified unprecedented heterogeneity of the mature neutrophil compartment in healthy mice. Maturation state and tissue origin were both highly associated with neutrophil diversity. Moreover, we found that transcriptional profiles of discrete neutrophil clusters were enriched in genes associated with specific functions or disease states, These results identified preexisting heterogeneity in neutrophils under steady state and suggest that a wide array of ready-to-use functional programs regulate neutrophil function both in health and disease. For single cell analysis of neutrophils, 8 weeks old and 20 months old C57BL6 mice were sacrificed, the lung, liver, spleen and intestine were dissected and dissociated into a single-cell suspension by enzyme digestion. Blood and bone marrow were colected either from the heart of from the femur. Cell strainer-filtered single-cell solution were sorted in BD Aria Cell Sorter as DAPI-CD11b+Ly6G+ cells and loaded into a BD Rhapsody cartridge. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to manufacture instructions. The experiment was perfromed twice and samples were multiplexed and loaded in two different BD cartridges for single cell library generation,