SUPPLEMENTARY FIGURES
Acute and sustained alterations to the bone marrow immune microenvironment following polymicrobial
infection
Dong Seong Cho1, Rebecca E Schmitt1, Aneesha Dasgupta1, Alexandra M Ducharme1, Jason D Doles1*
1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, 55905 USA.
*Corresponding Author:
Jason D Doles
Department of Biochemistry and Molecular Biology Mayo Clinic
200 First St SW
Guggenheim 16-11A1 Rochester, MN 55905 Tel: (507) 284-9372 Fax: (507) 284-3383
E-mail: [email protected]
Supplementary Figure S1. Single-cell RNA-sequencing (scRNA-seq) analysis on bone marrow cells. (A) Gating strategy to isolate propidium iodideneg single cells from cells isolated from mouse bone marrow by fluorescence activated cell sorting (FACS). (B) Uniform manifold
approximation and projection (UMAP) of bone marrow cells clustered into 32 clusters.
A B
Supplementary Figure S2.
Cluster 0, 1, 3, 4, 11, 12, 15, 17, 19, 20, 22, 25, 28, 29, 30: Erythrocyte
Cluster 2: Neutrophil
Cluster 5: T cell
Cluster 6: Erythroid cell Cluster 7: Erythroid cell
Cluster 9: Erythroid cell Cluster 31: Erythroid cell
Erythroblast-specific markers Cluster 6
Cluster 7
Cluster 9
Cluster 31
Cluster 8: Macrophage Cluster 10: Neutrophil
Supplementary Figure S2. (cont)
Cluster 13: Dendritic cell Cluster 14: Neutrophil
Cluster 16: Neutrophil
Cluster 21
Cluster 26 Eosinophil markers
(Ly6g-/Pglyrp1+)
Cluster 23: Neutrophil Cluster 24: Neutrophil
Supplementary Figure S2. Classification of 32 clusters of bone marrow cells into specific cell types based on differentially expressed genes (DEGs). Specific cell type markers that are differentially expressed in each cluster was shown with UMAP projections. Expression levels of the markers were represented by colors, and grey level UMAP highlights the cells of a given cluster.
Cluster 27: Plasmacytoid dendritic cell
Supplementary Figure S3.
Cluster 5: T cell
Sub-cluster 0: Basophil
Sub-cluster 1: Neutrophil
Sub-cluster 2: NK cell Sub-cluster 4: T memory cell
Sub-cluster 7: T memory cell Sub-cluster 8: NK cell
Sub-cluster 3: Long-term HSC (Gnl3 high)
Sub-cluster 6: Short-term HSC (Cd34 high)
Sub-cluster 5: Short-term Hematopoietic stem cell (HSC) (Cd34 high HSC)
Supplementary Figure S3. Sub-classification of cluster 5 cells into basophil, neutrophil, NK cell, T memory cell, short-term HSC and long- term HSC by sub-clustering. After clusters 5 was classified into T cell, they were sub-classified into basophil, neutrophil, NK cell, T memory cell, short- term HSC and long-term HSC based on DEGs between these clusters.
Supplementary Figure S4.
Supplementary Figure S4: Sub-classification of cluster 18 cells into 2 different cell types by sub-clustering. (A) Expression of B cell markers in cluster 18 is heterogeneous. Arrows shows subsets of cluster 18 where the markers are partially expressed in the cluster. (B) Cluster 18 cells were sub-
classified into pre-B cell and plasma cell based on DEGs between these clusters.
A
Sub-cluster 0: Igll1 and Vpreb1: Pre-B cell markers
Sub-cluster 1 & 2: Plasma cells
B
A B
Supplementary Figure S5: Gating strategies. Strategy to isolate propidium iodideneg single cells from cells isolated from mouse bone marrow 1 day post sepsis (A) and 1 month post sepsis (B).
Supplementary Figure S6.
Supplementary Figure S6: UMAP projection of bone marrow cells 1 day post sepsis. Classification of cell types in control was used to classify bone marrow cells 1 day post sepsis.
Supplementary Figure S7: Sub-classification of cluster 18 cells 1 day post sepsis into pre-B cell, plasma cell, and naïve B cell. Cluster 18 cells were sub-classified into pre-B cell, plasma cell, and naïve B cell by sub-
clustering within cluster 18.
Sub-clustering of cluster 18 (1D)
Sub-cluster 0: Plasma cell
Sub-cluster 1: Pre-B cell
Sub-cluster 2: Naïve B cell
Supplementary Figure S8.
Supplementary Figure S8: UMAP projection of bone marrow cells 1 month post sepsis. Classification of cell types in control was used to classify bone marrow cells 1 month post sepsis.
Supplementary Figure S9: Sub-classification of cluster 21 cells 1 month post sepsis into dendritic cell and eosinophil. Cluster 21 cells were sub- classified into dendritic cell and eosinophil by sub-clustering.
Sub-clustering of cluster 21
Dendritic cell markers (sub-clusters 1 and 3)
Eosinophil markers (sub-clusters 0, 2, and 4)
Supplementary Figure S10.
Control
1D
Supplementary Figure S10: Expression of apoptosis-associated markers in each cell type in control and 1D and 1M post-sepsis.
