Supplementary figures

Supplementary Figure 1: Detailed workflow of atlas-scale GRN analysis. To effectively integrate single-cell annotations across three atlases, we first manually devised 7 reference cell groups, chose TM-10x as a common reference and assigned the 55 cell types to 7 cell groups respectively. Next, we used scMAP to link each atlas (TM-SS2 and MCA) to the reference and built an integrated mouse atlas with common vocabulary for all single-cells. Using a stringent feature selection cutoff, we performed regulatory network inference using SCENIC. This briefly includes TF and TF-target identification from single-cell expression matrices (GRNBoost), cross-validation of TF and its direct targets (i.e. Regulons) using annotated motif databases and pruning away indirect, co-expressed (RCisTargets) and lastly scoring the regulon activity (RAS: regulon activity score) within each single-cell. We applied the framework to integrated and individual atlases to (i) classify individual and pseudobulk cells based global regulon activity (UMAP), (ii) classify cells based on shared and distinct regulon activity (cell-to-cell correlation), (iii) identify consensus and cell group specific regulons (regulon-to-regulon correlation) and (iv) build an atlas-scale regulon activity network.

Supplementary Figure 2: Cell type and cell group relationships A. Simplified mapping and projection of 55 reference cell types to 7 cell groups. B. scMAP visualisation of TM-10x cell atlas (reference onto itself) with 55 reference cell-types from 12 tissues.

Supplementary Figure 3: Mapping cell type annotations across individual cell atlases A. scMAP projection of TM-SS2 with 81 unique author assigned cell-type labels from 18 tissues to 55 reference cell types. B. scMAP projection of MCA with 732 unique author assigned cell-type labels from 38 tissues to 55 reference cell types.

Supplementary Figure 4: Mapping cell type annotations across integrated cell atlas A. scMAP projection of integrated mouse atlas with 831 unique author assigned cell-type labels from 50 tissues to 55 reference cell types.

Supplementary Figure 5: Examples of cell type annotations within individual tissues across integrated cell atlas. A. Projection of Liver cell types to reference cell groups across integrated cell atlases B. Projection of Spleen cell types to reference cell groups across integrated cell atlases

Supplementary Figure 6: Technical differences and biases in mouse cell atlases A. Number of cells (first row), library size (i.e. sequencing depth; second row) and number of genes detected (third row) for individual and integrated atlas. Each point represents a single- cell and the metrics are stratified and coloured based on 7 cell groups and 55 cell types with the median numbers reported next to box plots. B. Relationship between mean expression and dropout rate (percentage) for each atlas. TM- SS2 was more deeply sequenced and utilises full length Smart-seq2 protocol, while TM-10x and MCA apply 3’ end scRNA-seq protocols. C. Simplified table highlighting number of single- and pseudobulk cells, stratified by 7 cell groups across integrated cell atlas. D. Pie charts showing proportion of single- (top) and pseudobulk (bottom) cells across the 7 cell groups in individual and integrated cell atlas. Note: proportions are conserved between single- and pseudobulk cells.

Supplementary Figure 7: Technical differences and biases in cell atlases stratified by cell groups and reference cell types A. Number of cells (first row), library size (i.e. sequencing depth; second row) and number of genes detected (third row) for individual and integrated atlas stratified for each cell group and reference cell types. Although the 7 cell groups (and 55 reference cell groups within) are well represented from individual atlases; the Basal and Endothelial cells are under-represented in MCA.

Supplementary Figure 8: Tissue profiled across integrated mouse atlas and examples of regulon in single- and pseudobulk cells A. UMAP embedding of single-cells across integrated atlas based on RAS (same as Fig 1C), coloured by unique tissues across all cell atlases. B. UMAP embedding of pseudobulk across integrated atlas, based on RAS, coloured by unique tissues across all cell atlases. C. Examples of tissue- and reference cell-type specific regulons (Pou2af1, Cebpa, Gli1, E2f7 and ), coloured by RAS. D. Examples of general and cell-type specific regulons (Atf3, Elk3, Foxc1, Trp53), coloured by RAS.

Supplementary Figure 9: Cell type and tissue specific regulon across individual atlases. A. UMAP embedding of single cells in individual atlases coloured by reference cell types (top), RAS for immune Irf8 (middle) and cell type specific regulons (bottom: Sox17, and Pparg) B. UMAP embeddings of pseudobulk cells in individual atlases coloured by reference cell types (top), RAS of tissue-specific regulons (middle: Snai3 and bottom: Tcf7).

Supplementary Figure 10: Individual atlases stratified by tissues. UMAP embedding of (A) single-cells and (B) pseudobulk cells across individual cell atlas, coloured by unique tissue types.

Supplementary Figure 11: RAS comparison between single- and pseudobulk cells across integrated and individual atlases A. Distribution of Gini coefficients per regulon in pseudobulk and single-cells across integrated atlas, stratified by individual cell groups. B. Silhouette score comparing clustering between single- and pseudobulk cells across individual and integrated atlas. C. RAS correlation between single and pseudobulk cells in all cells and stratified by individual cell groups. Error bars represent the standard deviation across single- and pseudobulk cells. The individual cell group correlation is significantly improved compared to global, which further validates our classification of 7 cell groups.

Supplementary Figure 12: Impact of batch effect correction on regulon inference A. UMAP embedding of pseudobulk cells from Spleen in both TM-10x and TM-SS2 atlases, considering either uncorrected or two batch corrected expression space (BBKNN and MNN- correct). The pseudobulk cells are coloured by cell atlas (top) and cell groups (bottom). Note: Both batch correction methods slightly improve the overlap of reference cell types compared to uncorrected UMAP. However, the clusters from BBKNN and MNN-correct don’t overlap with each other and introduce additional discrepancies B. Pairwise correlation of individual regulons (based on RAS) from both batch correction methods compared to uncorrected data. Each dot represents a regulon identified in all 3 SCENIC runs (uncorrected, BBKNN and MNN-correct). The shaded area represents the 95% confidence interval from the linear regression line. C. Regulon composition similarity computed from pairwise Jaccard index between batch corrected (BBKNN and MNN-correct) to uncorrected data. The shaded area represents the 95% confidence interval from the linear regression line.

Supplementary Figure 13: Cell-type correlation across individual cell atlases A. Spearman correlation map of reference cell types within each cell atlas using pseudobulk cells. The first column highlights reference cell types. The cluster comparison between individual atlases and integrated atlases is presented in Fig. 1E. B. Pairwise correlation between individual cell atlases (‘TM-10x vs TM-SS2’, ‘TM-10x vs MCA’ and ‘TM-SS2 vs MCA’). The same colour scheme is used in A and B to indicate 55 reference cell types

Supplementary Figure 14: Regulon module features A. Significant (GO; biological processes) terms for each module. All regulons and direct targets within modules are used for GO analysis and are listed under the module (X- axis). The gene ratio highlights percentage of total GO term genes identified as enriched within the module. The gene list considering regulons alone (without direct targets) was too sparse to produce significant GO terms per module. B. Reactome pathway analysis using regulons within modules. Only regulons within modules are used for Pathway analysis and are listed under the module (X-axis). Only significant (qval<0.05) and enriched edges were considered for quantification and highlighted as tick sizes.

Supplementary Figure 15: Regulon correlation across individual cell atlases A-C. Connection specific index (CSI) matrix for each cell atlas. The identified regulons are coloured as in Fig 2B (5 modules), indicating co-regulatory and distinct roles across different cell types and tissues. Regulons identified in individual atlases but not in integrated atlas are marked in grey. D-F. Regulon network for each cell atlas. Each regulon is represented as a node, nodes are connected if CSI scores > 0.7 and coloured as in Fig 2C (5 modules). Both shared and unique regulon architecture are conserved in each cell atlas. Similar to integrated network (Fig. 2C), the larger modules (3 and 4) are bridged by smaller regulons (1, 2 and 5) within each cell atlas. The CSI for two nodes A and B is calculated by:

#*+,-. 0+**-01-, 1+ 2 +3 4 561ℎ 8!! ≥ 8!!$% − 0.05 !"#$% = 1 − *= The Pearson correlation coefficient (PCC) is the interactional correlation between A and B.

Supplementary Figure 16: Regulon activity in reference cell types across individual cell atlases. A-C. Individual regulon activities highlighted in regulon-by-cell type matrix for (A) TM-10x, (B) TM-SS2, and (C) MCA.

Supplementary Figure 17: Validation of regulon network A. Top: Motif correlation between individual regulons within each module. The rows and columns indicate individual motif sequences of different lengths. Bottom: representative examples of TFs and their enriched motifs for each regulon module. B. Annotated -protein interactions (PPi) from STRING overlaid on integrated regulon network. STRING contains all regulons (nodes), and only STRING validated interactions (black edges) are highlighted in the regulon network. 57% of regulon network edges are validated by STRING. C. Distribution of the STRING validated interactions captured in regulon network, plotted across 20 percentile combined score bins (x-axis). The number of regulon network links are listed above individual. The combined score is measure of confidence of STRING PPi. D. Correlation between regulon connection specificity index (CSI) and STRING confidence score. The error bars represent the 95% confidence interval. Red line indicates the CSI threshold used to construct regulon network. E. Regulon network overlaid with experimentally validated and essential genes (OGEE essentiality status). The enlarged nodes represent essential genes, while diminished nodes are annotated as non-essential. The regulons absent in OGEE are shown in ‘grey’.

Supplementary Figure 18, 19 and 20: Example for individual regulons composition and activity in reference cell types across cell atlases. Fig. S14. (A) Pou2af1, (B) Eomes and (C) Tcf7. Fig. S15. (A) Hsf1, (B) Etv3 and (C) Mafb. Fig. S16. (A) Irf5, (B) Irf9 and (C) Foxp1.

Supplementary Figure 21: Comparing different GRN methods for atlas-scale analysis A. Overlap of regulons and target genes identified in this study (SCENIC: GRNBoost and RCisTarget) and repeating analysis with published GRN method (bigSCale2; Ioconno et al 2015) for TM-10x mouse atlas. B. Jaccard index highlighting the overlap between regulon composition inferred on TM-10x atlas by our framework and repeating analysis with bigSCale2.

Supplementary Figure 22: Functional importance of regulon activity during myeloid differentiation A. UMAP embedding based on scRNA-seq expression from wildtype (multiple cell types) and Irf8 KO (or Irf8-/-) single-cells. B. UMAP embedding based on RAS from wildtype (multiple cell types) and Irf8 KO single- cells. The regulon space captures cell types differences as with expression space. C. Single-cell Irf8 expression in myeloid progenitors from wildtype (incl. monocytes, granulocytes) and Irf8 knockout cells (Purple). The Irf8 KO cells have a gradient expression of high and low cells. D. Irf8 regulon activity in myeloid progenitors from wildtype (incl. monocytes, granulocytes) and Irf8 KO cells (Purple). Note: the Irf8 KO cells have diminished RAS (compared to monocytes, consistent with change in their specification from monocyte to granulocytes. For calculating Irf8 regulon activity in both wildtype and Irf8 KO cells, we repeated AUCell 50 times and use the averaged activity score. E. UpSet plot of Irf8 regulon composition in wildtype monocytes, wildtype granulocytes and Irf8 KO cells. The Irf8 knockout cells alter and specify cell fate from monocytes to granulocytes, as highlighted by with drastically altered regulon composition in Irf8 KO cells.

Supplementary Figure 1 A. Detailed analyis workflow Tabula Muris Mouse cell Atlas

10x Smart-seq2 Microwell-seq (~54,000 cells) (~38,000 cells) (~150,000 cells)

Map cell types to a common reference 1. Manual annotation of atlas annotated cell types to 7 reference cell groups 2. TM-10x selected as reference. Assign 55 reference cell types to 7 reference cell groups 3. Use scMAP to map TM-SS2 and MCA author-annotated cell types to TM-10x (reference) (scMAP correlation threshold: 0.7)

Feature selection for regulon inference (Cutoff : Genes expressed in at least 10% cells)

Gene regulatory inference for integrated and each atlas (using SCENIC) 1. GRNBoost/GENIE3: Finds co-expressed and correlated TF-gene pairs 2. RCisTargets: Prunes away co-expressed TF-gene pairs using annotated motif database 3. AUCell: Scores regulons (TF-gene pairs) within each single-cell

Integrated TM-10x TM-SS2 MCA Tissues: 50 Tissues: 12 Tissues: 18 Tissues: 54

Single-cells (SC): 244,642 Single-cells (SC): 54,865 Single-cells (SC): 38,888 Single-cells (SC): 150,889 Pseudobulk (PB): 4,550 Pseudobulk (PB): 1,061 Pseudobulk (PB): 703 Pseudobulk (PB): 2,776

Cell types: 831 Cell types: 55 Cell types: 81 Cell types: 732 Features: 17,138 Features:19,369 Features: 21,373 Features: 34,967

Regulons: 174 Regulons: 222 Regulons: 233 Regulons: 233

Regulon activities (reference cell groups)

Cell-to-cell correlation

regulon-to-regulon correlation

Regulon activity network Supplementary Figure 2 A. reference cell types cell group (n=55) (n=7) Basal (3) Blood (3)

Stem (4)

Immune (22)

Epithelial (7)

Endothelial (4)

Specialised (12)

B. Author cell type Tissues reference cell types cell group (n=55) (n=12) (n=55) (n=7)

TM-10x Supplementary Figure 3

A. Author cell type Tissues reference cell types cell group (n=81) (n=18) (n=54) (n=7)

TM-SS2

B. Authors cell type Tissues reference cell types cell group (n=732) (n=38) (n=54) (n=7)

MCA Supplementary Figure 4 A. Cell atlases Author cell type Tissues Reference cell types Cell group (n=3) (n=831) (n=50) (n=55) (n=7)

MCA

TM-10x

TM-SS2 Supplementary Figure 5

Cell atlases Author cell type reference cell types cell group A. (n=3) (n=27) (n=36) (n=7)

Liver

Cell atlases Author cell type reference cell types cell group B. (n=3) (n=16) (n=34) (n=7)

Spleen Supplementary Figure 6 TM - 10X TM - SS2 MCA Integrated A. 80000

60000

Cells 40000

20000

0

106

4172 4521 4020 3033 14381 4877 8344 Cell groups 827 1108 1057 1020 1499 1119 956 Immune Specialised 104 580075905002881555557234713584775989792156 Stem

Library size Endothelial Basal Blood Epithelial 2 10 1250 2099 1368 6153 22892 3650 1751

105

2202 2215 2810 4499 695 1312 3328 3751 3871 1045 794 1037 104 1766 1467 1388 2914 1248 2538 1668 3192 912 517 652 659 653 865 598 613

103 Genes detected

102

Integrated mouse atlas cells B. 1.0 C. (by reference cell groups; also in Pie chat) Single-cell Pseudobulk 0.8 Basal 13394 261 Cell atlas Blood 2925 46 TM-10x 0.6 TM-SS2 Endothelial 8913 158 MCA Epithelial 30660 584 Immune 93461 1702 0.4 Specialised 27078 479

Dropout percentage Stem 68211 1310 0.2

0.0

0 2 4 6 log10(Mean Expression)

D. Single-cells TM - 10X TM - SS2 MCA Integrated 8.4% 6.8% 15.7% 10.0% 12.5% 2.8% 1.0% 40.7% 38.2% 14.4% 12.9% 13.2% 3.6% 36.7% 1.2% 30.6% 0.9% 0.6% 5.5% 0.3%

17.7% 15.2% 9.4% 27.9% 34.8% 11.1% 13.1% 14.6%

Pseudobulk TM - 10X TM - SS2 MCA Integrated 8.4% 6.7% 16.2% 10.0% 12.9% 2.8% 0.9% 40.0% 37.5% 3.5% 14.7% 13.9% 13.7% 36.3% 1.0% 29.3% 0.6% 5.7% 0.4% 0.3%

18.1% 15.8% 8.8% 28.9% 36.3% 10.6% 13.0% 13.9% Supplementary Figure 7 A. Immune Specialised

TM - 10X TM - SS2 MCA Integrated TM - 10X TM - SS2 MCA Integrated

20000 6000 15000 4000 Cells Cells 10000 2000 5000

0 0 107 107

106 106

105 105

104 104 Library size Library size 103 103

102 102 105 105

104 104

103 103 Genes detected Genes detected

102 102

Epithelial Endothelial

TM - 10X TM - SS2 MCA Integrated TM - 10X TM - SS2 MCA Integrated

15000 6000

10000 4000 Cells Cells

5000 2000

0 0 107 107

106 106

105 105

104 104 Library size Library size 103 103

102 102 105 105

104 104

103 103 Genes detected Genes detected

102 102

Basal Stem Blood TM - 10X TM - SS2 MCA Integrated TM - 10X TM - SS2 MCA Integrated TM - 10X TM - SS2 MCA Integrated 30000 2000 6000 25000 20000 1500 4000 15000

Cells Cells Cells 1000 10000 2000 500 5000 0 0 0 107 107 107 106 106 106 105 105 105 104 104 104 Library size Library size Library size 103 103 103 102 102 102 105 105 105

104 104 104

103 103 103 Genes detected Genes detected Genes detected

102 102 102 Supplementary Figure 8

A. Single-cells B. Pseudobulk

UMAP2 UMAP1 UMAP2 UMAP1

Bladder Kidney Marrow Fetal-Liver Peripheral_Blood Bone-Marrow Large_Intestine Mesenchymal-Stem-Cell-Cultured Fetal_Brain Placenta Bone-Marrow_c-kit Limb_Muscle Muscle Fetal_Intestine Prostate Bone_Marrow_Mesenchyme Liver Neonatal-Calvaria Fetal_Lung Skin Brain Lung Neonatal-Heart Fetal_Stomache Small-Intestine Brain_Myeloid MammaryGland.Involution Neonatal-Muscle Heart Thymus Brain_Non-Myeloid MammaryGland.Lactation Neonatal-Rib Heart_and_Aorta Tongue Embryonic-Mesenchyme MammaryGland.Pregnancy Spleen Neonatal-Skin Trachea Embryonic-Stem-Cell MammaryGland.Virgin Stomach Ovary Trophoblast-Stem-Cell Fat Mammary_Gland Testis Pancreas Uterus

C. D. Pou2af1 Cebpa Gli1 Atf3 Elk3

0 0.6 0 0.35 0 0.6 0 0.2 0 0.25 E2f7 E2f4 Foxc1 Trp53

0 0.4 0 0.25 0 0.55 0 0.12 Supplementary Figure 9

TM-10x TM-SS2 MCA A. (single-cells) (single-cells) (single-cells)

Immune Specialised Stem Endothelial Basal Blood Epithelial

UMAP2 UMAP1 Irf8 Irf8 Irf8

0 0.14 0 0.15 0 0.18 Sox17 Sox2 Pparg

0 0.55 0 0.30 0 0.27

TM-10x TM-SS2 MCA B. (Pseudobulk) (Pseudobulk) (Pseudobulk)

Immune Specialised Stem Endothelial Basal Blood Epithelial

UMAP2 UMAP1 Snai3 Snai3 Snai3

0 0.35 0 0.45 0 0.8 Tcf7 Tcf7 Tcf7

0 0.8 0 0.75 0 0.8 Supplementary Figure 10 A. TM-10x TM-SS2 MCA (single-cells) (single-cells) (single-cells)

B. TM-10x TM-SS2 MCA (Pseudobulk) (Pseudobulk) (Pseudobulk)

TM-10x tissues (12) TM-SS2 tissues (18) MCA tissues (39) Bladder Liver Ovary Bladder Lung Bladder Mammary_Gland Bone-Marrow Lung Pancreas Brain_Myeloid Mammary_Gland Heart_and_Aorta Marrow Bone-Marrow_c-kit MammaryGland.Involution Peripheral_Blood Brain_Non-Myeloid Marrow Kidney Spleen Bone_Marrow_Mesenchyme MammaryGland.Lactation Placenta Brain MammaryGland.Pregnancy Prostate Fat Pancreas Thymus Limb_Muscle Embryonic-Mesenchyme MammaryGland.Virgin Small-Intestine Heart Skin Liver Tongue Embryonic-Stem-Cell Mesenchymal-Stem Spleen Cell-Cultured Kidney Spleen Lung Trachea Fetal-Liver Muscle Stomach Large_Intestine Thymus Fetal_Brain Neonatal-Calvaria Testis Fetal_Intestine Neonatal-Heart Thymus Limb_Muscle Tongue Fetal_Lung Neonatal-Muscle Trophoblast- Stem-Cell Liver Trachea Fetal_Stomache Neonatal-Rib Uterus Kidney Neonatal-Skin Supplementary Figure 11 A. PB vs SC comparisons

All Celltypes Immune Epithelial cell Endothelial cell 1.0 1.0 1.0 1.0

0.8 0.8 0.8 0.8

0.6 0.6 0.6 0.6

0.4 0.4 0.4 0.4 Gini coefficient Gini coefficient Gini coefficient Gini coefficient 0.2 0.2 0.2 0.2

PB SC PB SC PB SC PB SC

Blood cell Stem cell Basal cell Specialized 1.0 1.0 1.0 1.0

0.8 0.8 0.8 0.8

0.6 0.6 0.6 0.6

0.4 0.4 0.4 Gini coefficient 0.4 Gini coefficient Gini coefficient Gini coefficient 0.2 0.2 0.2 0.2

PB SC PB SC PB SC PB SC

B. Single cell Pseudobulk 0.3 0.2

0.2 0.1 0.1 Silhouette score Silhouette score 0.0 0.0 TM - 10X TM - SS2 MCA Integrated TM - 10X TM - SS2 MCA Integrated

C. Integrated atlas (RAS correlation)

All Celltypes Immune Epithelial cell Endothelial cell 0.20 0.50 0.50 0.50 R (spearman): 0.18 R (spearman): 0.52 R (spearman): 0.33 R (spearman): 0.57 R (pearson): 0.07 R (pearson): 0.51 R (pearson): 0.38 R (pearson): 0.64

0.15

0.10 0.25 0.25 0.25 PB Mean RAS PB Mean RAS 0.05 PB Mean RAS PB Mean RAS

0.00 0.00 0.00 0.00 0.00 0.05 0.10 0.15 0.20 0.00 0.25 0.50 0.00 0.25 0.50 0.00 0.25 0.50 SC Mean RAS SC Mean RAS SC Mean RAS SC Mean RAS

Blood cell Stem cell Basal cell Specialized 0.50 0.50 0.50 0.50 R (spearman): 0.68 R (spearman): 0.3 R (spearman): 0.52 R (spearman): 0.39 R (pearson): 0.61 R (pearson): 0.26 R (pearson): 0.64 R (pearson): 0.35

0.25 0.25 0.25 0.25 PB Mean RAS PB Mean RAS PB Mean RAS PB Mean RAS

0.00 0.00 0.00 0.00 0.00 0.25 0.50 0.00 0.25 0.50 0.00 0.25 0.50 0.00 0.25 0.50 SC Mean RAS SC Mean RAS SC Mean RAS SC Mean RAS Supplementary Figure 12

A. Batch effect correction (expression space) No correction BBKNN MNN-correct

Cell atlas TM-10X TM-SS2 UMAP2 UMAP2

UMAP1

Cell groups Reference cell types Immune B cell endothelial cell of hepatic sinusoid luminal epithelial cell of mammary gland Specialised Stem DN1 thymic pro-T cell epithelial cell lung endothelial cell Endothelial Fraction A pre-pro B cell erythroblast macrophage Basal Blood Langerhans cell fibroblast mast cell Epithelial T cell granulocyte mesangial cell alveolar macrophage granulocytopoietic cell mesenchymal cell basal cell hematopoietic precursor cell mesenchymal stem cell basal cell of epidermis hepatocyte monocyte basophil immature B cell myeloid cell bladder cell immature T cell natural killer cell bladder urothelial cell keratinocyte neuroendocrine cell blood cell kidney capillary endothelial cell non-classical monocyte cardiac muscle cell kidney cell proerythroblast classical monocyte kidney collecting duct epithelial cell promonocyte dendritic cell kidney loop of Henle ascending limb epithelial cell skeletal muscle satellite cell early pro-B cell kidney proximal straight tubule epithelial cell stromal cell endocardial cell late pro-B cell type II pneumocyte endothelial cell leukocyte

B. RAS similarity C. Composition similarity to non-corrected data to non-corrected data 1.0 1.00 R (spearman): 0.64 0.8 0.75 0.6

0.4 0.50

0.2

MNN (jaccard index) 0.25 0.0 MNN (spearman correlation)

-0.2 0.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 BBKNN (spearman correlation) BBKNN (jaccard index) Supplementary Figure 13

A. Cell-to-cell (Pseudobulk) correlation within cell atlas TM-10x TM-SS2 MCA

Correlation Correlation Correlation 1 1 1

0 0 0

Immune Specialised Stem Endothelial Basal Blood Epithelial

B. Cell-to-cell (Pseudobulk) correlation between cell atlases MCA TM (10x) TM (10x)

TM (SS2) MCA TM (SS2) Correlation

0 1 Supplementary Figure 14

A. Gene Ontology

small GTPase mediated signal transduction positive regulation of cell adhesion Ras protein signal transduction regulation of binding negative regulation of phosphorylation ration rate adhesion negative regulation of protein phosphorylation positive regulation of catabolic process proteasomal protein catabolic process actin filament organization regulation of GTPase activity T cell activation regulation of apoptotic signaling pathway regulation of hemopoiesis regulation of protein catabolic process myeloid cell differentiation positive regulation of cellular catabolic process regulation of protein complex assembly GeneRatio regulation of protein serine/threonine kinase activity gland development epithelial tube morphogenesis 0.03 T cell differentiation Wnt signaling pathway 0.04 by wnt morphogenesis of a branching structure morphogenesis of a branching epithelium 0.05 embryonic organ development epithelial cell proliferation lymphocyte differentiation 0.06 cell junction organization skin development 0.07 mesenchyme development tube formation regulation of animal organ morphogenesis epithelial cell migration epithelium migration p.adjust autophagy process utilizing autophagic mechanism Golgi vesicle transport response to oxidative stress establishment of protein localization to organelle mitochondrion organization regulation of protein stability endomembrane system organization mRNA processing ribonucleoprotein complex biogenesis regulation of mitotic cell cycle nucleocytoplasmic transport nuclear transport mitotic cell cycle phase transition cell cycle phase transition RNA splicing DNA repair ncRNA metabolic process in utero embryonic development positive regulation of kinase activity regulation of vasculature development positive regulation of protein kinase activity positive regulation of cell projection organization regulation of angiogenesis axon development negative regulation of nervous system development negative regulation of neurogenesis negative regulation of cell development axonogenesis positive regulation of cell cycle Module 1 Module 2 Module 3 Module 4 Module 5 (7165) (605) (8655) (9286) (3344)

B. Pathway analysis (Reactome)

Transport of small molecules Signal Transduction Reproduction Programmed Cell Death Organelle biogenesis and maintainence Neuronal System Metabolism of Metabolism Immune System Homeostasis (Transcription) Disease Digestion and absorption Developmental Biology DNA Replication DNA Repair Circadian Clock Chromatin Organisation Cellular response to external stimuli Cell-Cell communication Cell Cycle Significant terms Module 1 Module 2 Module 3 Module 4 Module 5 0 46 (19) (2) (66) (74) (13) Supplementary Figure 15

A. TM-10x D.

E2f6 126g Sox11Klf13 43g 88g Foxo1 Nr1h2Tead4 40g21g Pbx3 Sox9 MafkKlf10 Taf7 Nr1h2 Klf7Gata2 396g 1052g Vezf1 Sox17Taf7 62g 51g Mef2aTal1 317g 165g MafgMax Msx1 Sox17 Tal1 Irf9Elk3 957g 2211g Nfat5 Foxc1 Klf7 PpargEts2 723g 68g Nfkb1Nfil3 436g 761g Atf4 CebpbTfe3 52g 1371g Fosl2 Irf1 Sox11 E2f6 MitfTcf7l2 745g 11g Gli3 Nfatc1Nfatc2 Hoxc10Hoxa10 68g93g Tcf12 Tead4 Hoxc6Tead3 25g57g Lhx2 Gata2 Elk3 Nr2f1Fhl2 17g 30g Tfe3Maff Rela Sox10Pou3f1 85g 8g Meis2 SrfBhlhe41 116g 138g Ddit3 Mef2a MaxFoxo1 84g 603g Nfia Fosb Foxp4Tcf12 59g133g Klf9 Tgif1 Stat3 Creb3l1 361g Junb NfiaTwist2 116g 140g Prrx2 Bcl3 Irf9 NficNfatc4 122g 548g Egr1 Gli2Gata6 4g 5g Fhl2 Jund Jun Ets2 Gli3Ddit3 19g 5g Cebpb Klf13 MafgPrrx2 190g38g Gata6 Sox9 56g Foxp4 Lhx2Vezf1 22g 257g Pknox1 Foxo3 Nfatc2Nfatc1 214g508g Creb3l1 FosbPbx3 559g71g Irf7 Nfat5Foxc1 12g 414g Fos Meis2 18g Twist2 Atf3 Pparg Fli1 Tgif1Tead2 324g 521g Nfic Tbx3 Msx1Pknox1 130g 17g Cebpe Klf1 Irf1Rela 807g 316g Cebpd Tead2 Stat3Bcl3 564g 386g Nfatc4 Nfkb1 Nfil3 Hdac2 Tfdp1 JunFos 540g866g Gli2 Nfe2 E2f7 Klf4Atf3 1061g1024g CebpdMyc 521g 637g Egr2 Bach1 Klf9Egr1 599g 1048g Nr2f1 Rfxap Arid3a Gtf2b Fosl2Klf10 418g999g Nr1d1 Rarg Atf4Maff 1142g840g Foxk1 Nfyb Egr2Mafk 32g567g Zbtb33 Crem Relb JundJunb 855g859g Dbp Sox10 E2f4 RfxapKlf16 105g 13g Fosl1 Elk1 Nfya RargTbx3 13g5g Gfi1 Pitx2Pitx1 20g55g Pitx2 Jdp2 Stat6 Nfkb2 Sox2Foxp2 41g 38g Xbp1 Zbtb17 Gmeb1 Grhl1Fosl1 371g998g Gli1 Irf5 Trp53 Patz1 EhfKlf5 435g906g Foxp2 Etv3 Irf8 Zbtb17Elf3 658g 13g Klf16 Tcf7l2 Usf2 MafbMxi1 199g669g Tead3 Rara Taf1 Zbtb7bCebpa 553g9g Hoxb2 Creb3 Rel Hcfc1 Hdac1 Hoxa7Creb3 70g27g Sox2Pitx1 Mafb Mxd1 Runx2 Tcf7 Sp3 Hoxb2Tbx2 848g 17g Sin3a Hoxb5Hoxa5 7g15g Hoxa10 Hoxa5 Elf4 Lef1 Ctcf Xbp1Nr2f2 1432g9g Pou3f1 Stat1 Gabpa DbpNr1d1 36g 42g Elf3 Foxj2 Nr6a1Gli1 80g 5g Klf5 Zbtb7b Usf1 Hoxa9Zbtb33 59g92g Nr2f2 Hoxc6 Batf Stat4 Cebpz Irf3 Hoxb8Hoxb7 17g40g Tcf3 Nr1h3 606g Srf Grhl1 Ikzf1 Klf15Tef 166g 896g Ehf Mxi1 Runx3 Pml PpardFoxa2 129g62g Mitf Stat5a Snai3 Spib Elf2 Srebf1Rxra 1251g 413g Hoxb5 Rest Brf1 Hnf1bHlf 8g 19g Hoxc10 Bhlhe41 Ets1 Irf2 Nr2f6Esrra 718g460g Cebpa Stat5b Klf1Hdac2 178g 13g Elf1 Foxp1 Stat5bStat5a 29g15g Tbx2 Elk4 Yy1 BatfCrem 300g 917g Nr6a1 Ppard Eomes Nr2c2 RelbNfkb2 783g 639g Gata3 Bach1Irf7 1162g 655g Hoxa7 Gtf2i Kdm5a Rfxank Etv3Jdp2 378g215g Setdb1 Sp1 Foxj2Rara 147g61g Nr1h3 Hoxb8 Srebf1 Bptf RelFoxk1 553g 16g Ep300 Irf8Irf5 1648g943g Nr3c1 Stat6Elf4 1152g 83g Klf15 Nr2f6 Hoxa9 Sp4 Pou2f1 Usf2Runx2 767g 306g Nfatc3 Arid3aMxd1 5g 86g Pou2af1 Nfe2Cebpe 531g 332g Hmga1 Pura Rfx1 Rxrb Foxo3Elk1 143g 78g Hoxb7 Esrra Rxra Bdp1 Sp100 Hmga1Fli1 1994g 6g E2f7Tfcp2 457g 6g E2f1Nfyb 631g1019g Gmeb2 E2f2Tfdp1 1301g 121g Tef Hnf1b Mef2d E2f4Gfi1 109g1641g Atf7 Gtf2bTrp53 165g336g Tfcp2 PuraNfya 260g13g Mecp2 Mecp2Gmeb2 6g 96g Foxa2 Nr3c1Nr2c2 503g279g RfxankAtf7 122g 53g Hlf Hsf1 BptfSp1 225g12g Sp100Sp4 94g 5g PmlFoxp1 557g 45g SpibIrf2 1027g 918g Elf2Usf1 251g 331g Yy1Elk4 326g301g Bdp1Ep300 183g 36g Irf3Kdm5a 206g 479g RestSetdb1 184g 260g Pou2af1Tcf3 31g 60g Patz1Pou2f1 10g 15g Hdac1Brf1 24g 13g Gmeb1Sp3 122g 93g Hcfc1Taf1 695g 1218g GabpaCtcf 55g 364g Hsf1Sin3a 11g 224g RxrbMef2d 13g 21g Rfx1Nfatc3 8g 4g Hlf 8g Eomes 303g

Gli2 4g Runx3 440g Rfx1 8g Gtf2i 9g Tbx3 5g Stat4 7g Sp1 12g Sp4 94g Srf 116g Gli1 80g Gli3 19g Stat4 7g Ctcf 55g Brf1 24g Tcf7 18g Max 84g Tcf3 31g Lef1 44g Taf7 62g Rel 553g Nr2f2 9g Fhl2 17g Ddit3 5g Tef 166g Tfe3 52g Dbp 36g Tfcp2 6g Mxd1 5g Ehf 435g Hsf1 11g Irf9 957g Irf5 943g Irf3 206g Irf1 807g Egr2 32g Pura 13g

Rxrb 13g Ikzf1 1043g Rarg 13g Gata6 5g Fos 866g Nr6a1 5g Jun 540g Lhx2 22g Sox2 41g Sox9 56g Yy1 326g Elf3 658g Elf2 251g Pitx1 55g Pitx2 20g Pbx3 71g Sp100 5g Sp3 122g Klf5 906g Klf13 43g Klf7 396g Klf9 599g Klf1 178g Pml 557g Nfic 122g Stat6 83g Batf 300g Prrx2 38g Mitf 745g Nfia 116g Gfi1 109g Nfatc3 4g Elk1 143g Elk4 301g Hoxb5 7g Myc 521g

Tal1 317g Stat1 1003g Ets2 723g Bptf 225g Bcl3 564g Foxj2 61g Atf7 122g Tcf12 59g Rela 316g Snai3 52g Nfat5 12g Rest 184g Patz1 10g Taf1 695g Nr2f1 30g Mecp2 6g E2f6 126g E2f7 457g Etv3 378g Rara 147g Rfxap 13g Relb 783g Usf2 767g Usf1 331g Spib 918g Maff 840g Irf7 1162g Irf8 1648g Irf2 1027g Nfya 260g Pou3f1 8g Nfil3 436g Meis2 18g Gata3 68g Pparg 68g Nfe2 531g Foxk1 16g Creb3 70g Foxa2 62g Fli1 1994g Tcf7l2 11g Nfyb 631g Fosb 559g Mxi1 199g Hmga1 6g Jdp2 215g Sox11 88g Sox17 51g Sox10 85g Esrra 718g Jund 855g Tbx2 848g Tead4 21g Tead3 57g Junb 859g Elf1 1530g Elf4 1152g Mafk 567g Foxo3 78g Nr1h2 40g Nr1d1 42g

Foxp2 38g Hnf1b 19g Foxp1 45g Cebpz 132g Zbtb7b 9g Ep300 36g Klf15 896g Tgif1 324g Klf10 418g Klf16 105g Klf4 1061g Hoxc6 25g Stat5a 15g Mafg 190g Hoxa7 27g Hoxa5 15g Hoxa9 59g Mafb 669g Hdac2 13g Hdac1 13g Stat3 386g Mef2d 21g Msx1 130g Fosl2 999g Fosl1 998g Bdp1 183g Stat5b 29g Arid3a 86g Elk3 2211g Hoxb2 17g Hoxb7 40g Hoxb8 17g Crem 917g Ets1 2036g Atf4 1142g Atf3 1024g Grhl1 371g Sin3a 224g Rfxank 53g Vezf1 257g Nr2f6 460g Rxra 1251g E2f1 1019g E2f4 1641g E2f2 1301g Gtf2b 165g

Egr1 1048g Elf1 1530g Trp53 336g Tfdp1 121g Pou2f1 15g Ikzf1 1043g Nr2c2 279g Nr3c1 503g Foxc1 414g Ppard 129g Nfkb1 761g Nfkb2 639g Zbtb17 13g Zbtb33 92g Bach1 655g Gmeb2 96g Gmeb1 93g Tead2 521g Foxo1 603g Nr1h3 606g Pknox1 17g Foxp4 133g Mef2a 165g Runx3 440g Runx2 306g Hoxc10 68g Cebpz 132g Hoxa10 93g Stat1 1003g Srebf1 413g Cebpa 553g Twist2 140g Cebpe 332g Nfatc4 548g Nfatc1 508g Nfatc2 214g Xbp1 1432g

Gabpa 364g Ets1 2036g Eomes 303g Cebpd 637g Hcfc1 1218g Pou2af1 60g Kdm5a 479g Gata2 1052g Setdb1 260g Gata3 68g Creb3l1 361g Cebpb 1371g Bhlhe41 138g Snai3Gtf2i 9g 52g Lef1Tcf7 18g44g

B. TM-SS2 E. Prrx2Twist2 Nfatc2Irf7 860g 8g Nfatc4 Nfkb1Ddit3 80g350g Hand2 Runx1Bach1 296g347g Cebpd Hoxa9Hlf 143g 15g Nfia Usf2Prdm1 329g 62g Twist1Gli2 Etv5Mef2a 224g 248g Six1 CebpaTfe3 513g 107g Rora MafgMafb 162g25g Nr1h3 Hoxc10 Nfe2Cebpe 64g 531g Usf1Ep300 612g 633g Foxp2 Pbx1 Cebpz 115g Nfic Egr2 Irf1 Fli1Rxrb 1640g 10g Plagl1 Stat1Irf9 755g 867g RelRunx2 681g 515g Tcf7l1 Irf2Runx3 731g 586g Sox5Gli1Nr2f2Xbp1 Irf8Irf5 1306g961g Cebpb Elf4Arid3a 893g 150g Junb BatfIkzf1 29g 1230g RaraEtv3 366g134g Nfix Elk1Setdb1 220g 336g Nr2f1Klf9Msx1 E2f6Taf1 902g721g Meox1 Hdac2Arntl 31g 5g Hoxb2 Rbpj Hmgb2Rb1 53g 10g Gfi1E2f7 243g415g Egr1 E2f2Tfdp1 865g 997g Fosb Mitf NfyaE2f1 192g809g Atf4 Stat3 Jdp2 NfybCux1 290g 166g Trp53E2f4 1222g 418g Jund Fosl2 Stat5aEomes 81g101g Stat5bLef1 11g 21g Hoxc6 Mef2dTcf7 17g 9g Myc Atf7 Nr3c1 RestBrf1 175g169g Creb3l1 Hsf1 Kdm5aYy1 420g 624g Bcl3 Foxp1Sp3 128g 6g Tcf3Pou2af1 9g 65g Nfil3 Elf1Foxo1 1686g 310g Maff SpibPml 516g 701g Hcfc1Gtf2b 129g1019g Sox11 Nfkb1 CtcfNr2c2 243g 612g Relb Cnot3Sin3a 576g54g Runx1 Zfp668Creb1 111g 9g Rfx2 Crem Pou2f1Irf3 213g 12g Mafk Bach1 Nfatc2 Sp1Ets1 238g1508g Tbx2 Tgif1 GabpaTaf7 135g 585g Elk4Elf2 729g459g Hoxa5 Nfat5Gmeb1 10g 19g Hoxb5 Ddit3 Mafb Gmeb2Nfkb2 466g 114g Klf4 Klf15 Bdp1Nr1h2 180g 8g CebpaEtv3 PuraPatz1 9g 13g Foxc1 Rela Hsf1Mnt 10g 11g Fosl1 Tcf12Foxk1 4g4g Klf10 Nfat5 Irf5 Tead2Srf 148g 126g Tfe3 Gata2Sox17 758g231g Fos Erf Etv5 Irf8 Elk3Klf7 197g1344g Mafg Elf4 Ets2Tal1 184g690g Atf3 Tcf4 Tal1 Rxrb ErfTead4 91g 162g Gata6 Tead3 Rara Klf15Pknox1 444g 9g Prdm1Mef2a Ikzf1 Meis2Dbp 477g 79g Gata2 Arid3a Sox10Srebf1 714g409g Klf16 Creb3 ClockSox2 690g144g Jun Irf7 Foxj2Tef 213g 299g Sox17 Fli1 Nfe2Cebpe Atf2Mecp2 100g 203g Gata3 Dbp Pbx3Zbtb33 15g 132g Mecom Klf7 Batf Bhlhe41Sp4 12g 820g Rarg Elk3 Irf2 Runx2 Mxi1Creb3 302g 416g Nr1h2 Usf1 Hoxb4Stat6 15g 20g Hoxa7 Ahr Irf9 Zbtb7bAhr 21g 279g Tcf7l2 Tead4 JunNr1d1 794g 31g Ets2 Usf2 FosAtf3 827g723g Barx2Nr1d1 Bhlhe41 Stat1 Rel Tcf7l2Klf4 720g 535g Sox10 Hlf Foxo3Hoxb7 131g86g Grhl1 Mxi1 Runx3 Ovol2Klf5 1371g 7g Snai3 Nfe2l1Foxq1 38g14g Hoxb4 Arnt Hoxa9 Grhl1Hoxa7 346g 24g Rxra Gata3Rxra 470g 112g Zbtb7b Stat6 RargBarx2 41g 34g Foxq1 Nfkb2 Snai3Hif1a 193g7g Hif1a Sox2 Cebpz E2f2E2f1 MecomGata6 29g 8g Nfe2l1Klf1 Meis2Tead2 Ep300 Klf1Foxa2 19g 27g Ovol2Foxo3 Tcf12 Pml Foxp1 Etv4Pparg 8g 208g Nfya Hnf1bMlx 19g 734g Klf5 Spib Zfp668 Nr2f6Esrra 681g591g Elf3 Foxj2 Eomes Creb3l4Ppard 395g 51g Pparg Pou2af1 E2f7 EhfElf3 285g257g Hoxb7 ClockTef Rb1 Bcl3Pitx2 659g 40g Atf2 Hmgb2 Tgif1Stat3 409g578g Pitx2 Nr2f6 Srebf1 Pbx3 Ets1 Sin3a Fosl1Klf16 162g1149g Ehf Patz1 Gmeb2 Elf1 Tcf3 Klf10Tead3 302g 10g Ppard Pknox1 Mecp2 Foxo1 Gfi1 MafkMaff 361g398g Mlx Foxa2 Srf Taf7 Elk4 Hcfc1 RelaCrem 131g 530g Creb3l4 Etv4 E2f6 ArntRelb 149g427g Sp4 Zbtb33 Setdb1 Pou2f1 Tfdp1 Nr3c1Atf7 51g 13g Esrra Bdp1 Sp1 E2f4 Hoxa5Sox11 212g132g Irf3 Tbx2Hoxb5 8g 137g Hnf1b Gmeb1 Nr2c2 Gtf2b MitfTcf4 697g 278g Pura Elf2 Ctcf CebpbIrf1 712g 1078g Arntl RbpjJdp2 10g153g Yy1 Trp53 Hdac2 Tcf7l1Creb3l1 213g 853g Meox1Xbp1 995g 196g Elk1 Kdm5a Nr2f2Msx1 164g269g Taf1 Stat5aRest Gli2Plagl1 19g 118g Sp3 Nfyb CebpdHoxc10 673g 110g Cnot3 Klf9Six1 394g125g Lef1Cux1 Gli1Rora 26g 10g Brf1 Nr1h3Pbx1 19g 417g Gabpa NficFoxp2 10g 6g Hand2Sox5 11g 30g Stat5b Nfatc4Prrx2 16g 392g Foxk1 Mef2dCreb1 Twist2Twist1 214g241g Tcf7 Erf 91g Nfia 8g Tcf3 9g Etv4 8g

Pura 9g Nfia 8g Ahr 21g Mlx 19g Tbx2 8g Sp4 12g Klf1 19g Rb1 53g Srf 148g Gli2 19g Gli1 26g Nfic 10g Batf 29g Hlf 143g Mnt 10g Tcf7 17g Atf7 51g Tcf12 4g Snai3 7g Lef1 11g Rel 681g Nr2f1 8g Tef 213g Rfx2 10g Nr2f1 8g Ehf 285g Hsf1 11g Irf7 860g Irf9 755g Irf2 731g Irf5 961g Irf3 213g Irf1 712g Rxrb 10g Egr1 58g Rora 10g Rarg 41g Rbpj 10g Fos 827g Ovol2 7g Nfe2 64g Foxk1 4g Jun 794g Sox5 11g Yy1 420g Elf4 893g Elf2 729g Elf3 257g Nr1h2 8g Arntl 31g Foxp1 6g Foxp2 6g Pitx2 40g Pbx3 15g Pbx1 19g Sp3 128g Sp1 238g Klf7 197g Klf4 720g Klf9 394g Pml 516g Mafb 25g Hdac2 5g Stat6 15g Nfix 195g Mef2d 9g Ctcf 243g Prrx2 16g Mitf 697g Brf1 175g Gfi1 243g Nfatc2 8g Six1 125g Elk1 220g Elk4 459g Myc 858g Tal1 184g

Ets2 690g Rfx2 10g Bcl3 659g Atf2 100g Atf3 723g Tcf4 278g Rela 131g Nfat5 10g Rest 169g Patz1 13g Taf1 902g Taf7 135g Ddit3 80g Tfe3 513g Dbp 477g E2f6 721g E2f7 415g E2f2 865g E2f1 809g Rxra 470g Etv5 224g Etv3 366g Barx2 34g Rara 134g Arnt 149g Usf2 329g Usf1 612g Relb 427g Spib 701g Maff 361g Irf8 1306g Egr2 169g Nfya 192g Zfp668 9g Nfil3 480g Meis2 79g Gata6 29g Nr3c1 13g Fli1 1640g Foxa2 27g Nfyb 290g Fosb 598g Cnot3 54g Mxi1 302g Jdp2 153g Sox2 690g Esrra 681g Jund 778g Elf1 1686g Tead3 10g Junb 863g Mafk 398g Pknox1 9g Nr1d1 31g Foxq1 38g Klf15 444g Klf5 1371g Tgif1 409g Klf16 162g Klf10 302g Cux1 166g Mecom 8g Stat5a 81g

Mafg 162g Nfil3 480g Hoxa9 15g Stat1 867g Hoxa7 24g Stat3 578g Hif1a 193g Msx1 164g Fosl2 907g Bdp1 180g Nfe2l1 14g Stat5b 21g Elk3 1344g Xbp1 995g Hoxb4 20g Hoxb7 86g Crem 530g Ets1 1508g Prdm1 62g Foxj2 299g Hand2 30g Atf4 1024g Sin3a 576g Grhl1 346g Clock 144g Nr2f6 591g Nr2f2 269g E2f4 1222g Gtf2b 129g Trp53 418g Tfdp1 997g Pou2f1 12g Ikzf1 1230g Gata2 758g Gata3 112g Nr2c2 612g Foxc1 157g Pparg 208g Ppard 395g Creb1 111g Creb3 416g Nfkb1 350g Tcf7l2 535g Tcf7l1 213g Nfkb2 466g Sox17 231g Sox10 714g Sox11 212g Bach1 296g Fosl2 907g Gmeb1 19g Tead2 126g Tead4 162g Foxo1 310g Plagl1 118g Foxo3 131g Nr1h3 417g Hnf1b 734g Mef2a 248g Ep300 633g Hmgb2 10g Runx1 347g Runx2 515g Runx3 586g Hoxc6 177g Cebpz 115g Hoxa5 132g Srebf1 409g Fosl1 1149g Cebpa 107g Twist1 241g Twist2 214g Cebpe 531g Creb3l4 51g Nfatc4 392g Arid3a 150g Hoxb5 137g Gabpa 585g Hoxb2 162g Eomes 101g Cebpd 673g Meox1 196g Mecp2 203g Hcfc1 1019g Pou2af1 65g

Kdm5a 624g Jund 778g Setdb1 336g Zbtb33 132g Gmeb2 114g Zbtb7b 279g Hoxc10 110g Creb3l1 853g

Cebpb 1078g Atf4 1024g Bhlhe41 820g Myc 858g FosbEgr1 58g598g JunbEgr2 169g863g Hoxb2Foxc1 157g162g NfixHoxc6 195g 177g

C. MCA F. Rfxap RbpjCrem 6g 1623g Stat3Cebpb 320g 1054g Hivep1Prdm1 324g61g MafbTgif1 260g245g Ets2Ddit3 1197g 66g Hivep2Fosl2 808g 60g Foxo1Mef2a 649g19g JunbTfe3 119g1114g Nfil3Jund 375g27g RargMsx1 6g6g Nfe2l1Msx2 97g 8g Egr2Elk3 995g225g Elk1 Atf3Fosb 1855g 838g Hsf1 JunFos 1123g1115g Nfe2l1Pou3f1 Setdb1 MitfEgr1 202g 234g Sox2 Sox6Sox5 8g31g Nfat5Hif1a 238g10g Snai3 Elf5 Cebpz Fosl1Srf 73g 1058g Pbx3 Nfatc1Mafk 460g 400g Eomes Taf7 MaffAtf4 1099g623g Trp53 Sox9Myc 696g 117g Tead4 E2f4 Nfatc2Foxc1 91g162g Zbtb17 Klf1 Atf7Tead1 18g 417g Foxo3 Klf5 Nfyb NficAr 5g 11g Etv4 Gli3Gli2 25g14g EhfHmgb1 Creb3l1Twist1 30g 911g Foxp4 Ybx1 Ahr Tal1 NfiaTcf4 59g340g Rest Bdp1 Hoxc6Thra 97g 72g Sall2 Nfatc3 Nfatc4Gli1 24g 257g Hdac2 Vdr Hoxa10Pbx1 7g 45g Hoxb7 Nr2f6 Etv1Tcf7l1 160g 263g Hnf1b Esrra Zfpm1 DbpTef 114g 195g Tead2 Foxm1 E2f7Sin3a Creb3Bhlhe41 790g 158g Hoxb5 Klf15 Foxq1 Hoxa9Sox10 13g42g Hoxa7 NfixTbx3 46g 18g Mecom Atf2 Gata6Tbx2 8g 106g Sox11 Mlx Foxk1 Creb3l4 Cnot3Gmeb1Tfdp1 Nr2f2Tcf7l2 167g 42g Foxp2 Zbtb17Tead2 661g38g Hoxb8Hoxa5 Hoxb5Hoxa5 27g22g Tcf7l2 Ppard Elf3 E2f6 Sall2Hdac2 10g 151g Hoxc10Hoxb7 19g 45g Grhl1Gata3 Foxp2Tead3 73g115g Meis1 Hoxa7Sox11 509g25g Tead3 Gtf2b Hoxb2Hoxb8 29g75g Foxa2 Hlf Gabpa Hand2Meis1 40g 9g Tbx3 Msx2 Srebf1Pitx2 9g 658g E2f1 Usf2 HlfSox17 17g 51g Hoxb2Hoxa9 Xbp1Nr1h3 1945g 764g PpargPpard 403g370g Nr2f2 Hcfc1 Foxa2Foxp4 352g18g Pitx2Thra Hoxb4 Cux1 EhfMecom 1330g 10g Etv1 Yy1 Hnf1bNr2f6 441g204g Hand2 Bhlhe41 Pparg Elf3Klf15 1095g 718g Gfi1 Gata3Esrra 701g 248g Erf Gli1 Ovol2 Taf1 Stat5a Foxq1Grhl1 175g 26g Srebf1 MaxVdr 18g 3g Atf7 Hoxa10 Nfe2 Patz1 Gmeb2 Nr3c1Runx1 148g517g Sox10 Nr2c2 Elf4Ikzf1 828g 1415g Tbx2Hoxc10Gata6 E2f2 Klf13Fli1 1440g 13g Pbx1 Nfya Irf8Rara 2067g 7g Sox17 Rfx2 Cebpe Tcf12 Ets1Pou2af1 2229g 58g Sp1 Elf1Spib 2081g 881g Hoxc6 Nfix Dbp Tef Klf16Runx3 14g 474g Tcf7l1 Stat4 Bach2Foxp1 13g4g Nfatc4 Mxi1 Ctcf Bach1Batf 29g 20g Arnt Mecp2 Bcl3Rel 929g 322g Nfkb2Irf1 1828g 789g Xbp1 Gata2 Irf7Relb 1596g 496g Nfic Tcf4 Creb3 Arid3a Irf5Etv3 1163g 235g Nr1h3 PmlKdm5aBptf Irf9Irf2 1143g791g Usf1Stat1 287g 1795g Gtf2ird1 Cebpd Stat6 Mecp2Mef2d 209g90g BptfTcf12 187g 9g Ar Mitf Msx1 Sp3 RxrbKdm5a 50g 299g Nfia RelaStat5b Sp4Irf3 234g 117g Nr3c1 Lef1Tcf7 11g38g Mafg Elk4Elf2 317g301g E2f1Sp3 101g640g Twist1 Elf4 Usf1 Hcfc1Gfi1 185g 417g Creb3l1 Elk4 Elf2 Mxi1Arid3a 536g 151g Cebpa Stat4E2f2 707g6g Ikzf1 Nfe2Cebpe 100g 479g Gli3 Egr1Jun Ep300Pml 482g 7g Runx1 RelaStat6 112g 10g Gli2 Tead1 Elk3 Fli1 Elf1 Irf3Rxrb Usf2Cux1 10g 5g Atf3 Stat5bSp1 55g 69g Crem NfyaCtcf 353g76g FosbFos Mef2d Yy1Nr2c2 178g 164g Nfil3 Max Pou2af1 Patz1Gmeb2 3g 48g Rarg Tcf7 CebpdStat5a 108g1021g Egr2 Sox6 Irf8 Gata2Mafg 324g 505g RxraCebpa 491g 797g Hif1a Atf4 Runx3 Hmgb1Ahr 6g 5g Sox9 Ets1 Klf1Tal1 66g270g Sox5 Rxra Creb3l4Gtf2ird1 969g18g Nfatc2 Ets2 Irf5 Rfx2Ovol2 568g 116g Fosl1 Jund Cebpb SpibLef1 Ybx1Klf5 497g 540g Maff Klf13 Irf2 Setdb1Elk1 94g 185g Mef2a Mafk Irf7 E2f4Trp53 1358g 362g Fosl2 CebpzNfyb 559g 178g Myc Ddit3 Bach2Foxp1Sp4 Rest 143g Nfat5 Stat3 Etv3 Sox2Taf7 230g38g Nfatc1 Junb Prdm1 Batf Pou3f1Pbx3 46g 21g Foxc1 Bcl3 Bach1 EomesTead4 150g292g Foxo1 Hivep1Mafb Elf5Snai3 3260g 8g Tgif1 Rel Ep300 Foxo3Etv4 1008g 11g Hivep2 Irf9 Stat1 Pknox1Erf 6g 15g Srf Foxk1Rfxap 2g6g Nfkb2 Rara Zfpm1Mlx 5g 8g Relb Hoxb4Arnt 42g 7g Tfe3 Irf1 Ar 5g

Erf 6g E2f7 104g Ahr 6g Mlx 5g Tfdp1 744g Srf 73g Hlf 17g Max 3g Rara 7g Rarg 6g Rbpj 6g Vdr 18g Foxm1 4g Sox6 8g Tbx2 8g Pitx2 9g Pbx1 7g Sp1 55g Klf1 66g Cux1 5g Nfic 11g Gli2 14g Gli3 25g Gli1 24g Stat4 6g Nfix 46g Batf 29g Msx1 6g Nfia 59g Elk1 94g Atf7 18g Tcf12 9g Tcf7 11g Snai3 8g Patz1 3g Lef1 38g Rel 929g Tef 114g E2f6 68g Arnt 42g Rfxap 2g Usf2 10g Hsf1 15g Thra 97g Irf2 791g Irf3 234g Rxrb 50g

Nfya 76g Gmeb1 26g Sall2 10g Foxk1 6g Sox2 38g Sox5 31g Bach2 4g Yy1 178g Jund 27g Tbx3 18g Elf4 828g Elf2 317g Pbx3 46g Ep300 7g Sp4 117g Sp3 101g Klf5 497g Klf13 13g Klf16 14g Rbpj Pml 482g Stat6 10g Ctcf 353g Mitf 202g Msx2 97g Bdp1 26g Gfi1 185g Nfe2l1 8g Nfatc3 4g Elk4 301g Elk3 995g Hoxb4 7g Myc 696g Tal1 270g Bptf 187g Zfpm1 8g Bcl3 322g Hand2 9g Atf2 194g Tcf4 340g Rela 112g Nfat5 10g Rest 143g

Taf7 230g Atf2 194g Ddit3 66g Tfe3 119g Dbp 195g Rfx2 568g E2f2 707g Rxra 491g E2f7 104g E2f1 640g Etv3 235g Etv1 160g Foxm1 4g Relb 496g Usf1 287g Ehf 1330g Spib 881g Maff 623g Irf5 1163g Irf8 2067g Irf1 1828g Irf7 1596g Irf9 1143g Egr2 225g Egr1 234g Nfil3 375g Meis1 40g Fos 1115g Foxc1 91g Nfe2 100g Fli1 1440g Tcf7l2 42g Nfyb 559g Jun 1123g Fosb 838g Mxi1 536g Sox10 13g Sox17 51g Sox9 117g Esrra 701g Bach1 20g Elf5 3260g Elf3 1095g Elf1 2081g Foxo3 11g Mafk 460g Foxq1 26g Foxp2 73g Foxp1 13g Foxp4 18g Mef2a 19g Ybx1 540g Klf15 718g Tgif1 245g Hmgb1 5g Hoxc6 72g Mafg 324g Hoxa5 22g Hoxa9 42g Hoxa7 25g Mafb 260g Stat3 320g Hif1a 238g

Fosl2 808g Bdp1 26g Twist1 30g Stat5b 69g Hoxb5 27g Hoxb7 19g Hoxb8 75g Hoxb2 29g Ets1 2229g Ets2 1197g Atf3 1855g Atf4 1099g Sin3a 233g Grhl1 175g Taf1 1467g Nr2f6 441g Nr2f2 167g Mecp2 90g E2f4 1358g Etv4 1008g Gtf2b 168g Hcfc1 417g Trp53 362g Tfdp1 744g Pou3f1 21g Hivep1 61g Hivep2 60g Gata2 505g Gata3 248g Ikzf1 1415g Gata6 106g Nr2c2 164g Nr3c1 148g Ovol2 116g Pparg 403g Ppard 370g Creb3 790g Foxa2 352g Tcf7l1 263g Nfkb2 789g Cnot3 156g Sox11 509g Zbtb17 38g Gmeb2 48g Gmeb1 26g Tead4 150g Tead3 115g Tead2 661g Tead1 417g Junb 1114g Foxo1 649g Nr1h3 764g Pknox1 15g

Hnf1b 204g Hsf1 15g Runx1 517g Runx3 474g Mecom 10g Hoxc10 45g Stat5a 108g Cebpz 178g Hdac2 151g Hoxa10 45g Stat1 1795g Mef2d 209g Fosl1 1058g Srebf1 658g Cebpa 797g Cebpe 479g Nfatc1 400g Nfatc2 162g Nfatc4 257g Arid3a 151g Xbp1 1945g Gabpa 378g Crem 1623g Eomes 292g Prdm1 324g Gtf2ird1 18g Pou2af1 58g Kdm5a 299g

Setdb1 185g Nfatc3 4g Creb3l4 969g Creb3l1 911g Cebpd 1021g Cebpb 1054g Gabpa 378g Bhlhe41 158g Sin3a 233g Klf16 Gtf2bE2f6 68g 168g Cnot3Taf1 1467g 156g Supplementary Figure16 B. A. C. MCA TM (SS2) TM (10x)

Atf7 Tcf7l2 Gata6 Tfcp2 Sox17 Ovol2 Hoxa7 Etv1 Creb3l4 Hoxb7 Bhlhe41 Esrra Mitf Hoxc6 Tead3 Pparg Meis1 Hif1a Foxa2 Egr1 Gata3 Nr2f6 Gli3 Rarg Klf5 Nfix Atf3 Pitx1 Dbp Mafk Klf16 Hoxb5 Nfkb2 Bhlhe41 Tbx2 Irf1 Rfx1 Jun Gli1 Tbx3 Gli2 Foxp2 Irf1 Nfat5 Plagl1 Taf7 Fosl1 Klf7 Myc Fosb Mafg Foxc1 Cebpd Nfatc2 Foxp4 Twist1 Klf10 Ehf Foxc1 Dbp Foxq1 Hoxb2 Prrx2 Hoxb7 Tal1 Gli3 Hlf Klf7 Egr1 Esrra Hoxb4 Atf3 Elk1 Patz1 Fosb Cebpz Klf15 Snai3 Maff Trp53 Clock Crem Foxm1 Dbp Nfat5 Elf5 Gmeb2 Hoxb2 Sall2 Foxj2 Nr2f1 Pknox1 E2f6 Max Usf2 Gtf2b Cebpe Elk4 Rb1 Batf Ctcf E2f1 Jdp2 Usf1 Stat5a Relb Nr2c2 Zfp668 Irf7 Gtf2b Foxp1 Stat6 E2f1 Spib Irf2 Hcfc1 Taf7 Foxj2 Pou2af1 Eomes Mef2d Sin3a Kdm5a Elk4 Snai3 Mafg Sp1 Tcf7 Bdp1 Erf Hmga1 Elf4 Rxrb Patz1 Irf2 Usf1 Sp3 Yy1 Runx3 Mafb monocyte classical monocyte non-classical monocyte macrophage leukocyte myeloid cell granulocyte alveolar macrophage blood cell late pro-Bcell Fraction Apre-proBcell immature Tcell promonocyte granulocytopoietic cell basophil hematopoietic precursorcell erythroblast proerythroblast T cell natural killercell kidney collectingductepithelialcell kidney loopofHenleascendinglimbepithelialcell type IIpneumocyte kidney proximalstraighttubuleepithelialcell cardiac musclecell hepatocyte B cell immature Bcell early pro-Bcell epithelial cell bladder urothelialcell luminal epithelialcellofmammarygland basal cell basal cellofepidermis keratinocyte fibroblast stromal cell neuroendocrine cell mesangial cell bladder cell skeletal musclesatellitecell mesenchymal stemcell kidney capillaryendothelialcell endothelial cell lung endothelialcell endocardial cell mesenchymal cell Nfkb2 T cell skeletal musclesatellite cell hematopoietic precursor cell luminal epithelialcellof mammarygland basal cell late pro-Bcell proerythroblast natural killercell DN1 thymicpro-Tcell macrophage myeloid cell kidney cell immature Bcell B cell monocyte classical monocyte non-classical monocyte promonocyte dendritic cell alveolar macrophage blood cell granulocytopoietic cell granulocyte basophil leukocyte type IIpneumocyte kidney loopofHenleascendinglimbepithelialcell kidney collectingductepithelialcell kidney proximalstraighttubuleepithelialcell hepatocyte cardiac musclecell bladder urothelialcell mesangial cell neuroendocrine cell fibroblast bladder cell mesenchymal stemcell endothelial cell endocardial cell stromal cell early pro-Bcell immature Tcell Langerhans cell mesenchymal cell Creb1 E2f2 late pro-Bcell early pro-Bcell kidney collectingductepithelialcell basal cellofepidermis keratinocyte bladder urothelialcell fibroblast mesenchymal stemcell bladder cell mesenchymal cell Irf8 Pbx3 luminal epithelialcellofmammarygland epithelial cell type IIpneumocyte hepatocyte kidney proximalstraighttubuleepithelialcell cardiac musclecell neuroendocrine cell stromal cell mesangial cell kidney capillaryendothelialcell endothelial cell lung endothelialcell endothelial cellofhepaticsinusoid endocardial cell skeletal musclesatellitecell basal cell myeloid cell classical monocyte blood cell Langerhans cell kidney cell monocyte granulocyte leukocyte non-classical monocyte macrophage mast cell alveolar macrophage T cell DN1 thymicpro-Tcell natural killercell immature Tcell dendritic cell immature Bcell B cell granulocytopoietic cell Fraction Apre-proBcell hematopoietic precursorcell basophil Tfdp1 Bcl3 Ahr Trp53 Sp4 Irf9 Sp1 Cebpa Irf2 Pml Mafb Eomes Pou2af1 Hif1a Mxi1 Ep300 Jund Bptf Cux1 Jdp2 Atf2 Nfe2 Supplementary Figure 17

A. Overrepresented motifs per module Motif Correlation

0.0 1.0 Module 1 Module 2 Module 3 Module 4 Module 5 Regulon motif Regulon motif Regulon motif Regulon motif Regulon motif

Regulon motif Regulon motif Regulon motif Regulon motif Regulon motif

Bach1_MA0591.1_MM Gata3_PB0021.1_MM Hoxa5_PH0052.1_MM Irf8_MA0652.1_HS Snai3_MA1559.1_HS

Cebpa_MA0102.1_MM Klf16_MA0741.1_HS Jun_MA0488.1_HS Tcf7_MA0769.1_MM Atf7_MA0834.1_HS

Cebpd_MA0836.1_HS Hoxb5_MA0904.1_MM Irf5_MA1420.1_HS Pbx3_MA1114.1_HS

Mafb_MA0117.2_MM Fos_MA0476.1_HS Foxp1_MA0481.1_HS Pknox1_PH0140.1_MM

B. Protein-protein interactions (SPRING) C. GRN network links also predicted in STRING 738 0.3

536 0.2 394 342 286 0.1

0.0 Fraction (Regulon network) First Second Third Fourth Last (150-183) (183-224) (224-289) (289-426) (426-999) STRING link “Combined score” percentile

Total network edges: 4032 Edges identified in SPRING: 2290 (57%) D. Regulon activity (CSI) vs STRING confidence

420 Minimum threshold for Regulon network 400 E. Essential Genes (OGEE) 380 Tef 360 Hlf PpardNr2f6 Esrra Ehf Hoxa9 Foxa2Srebf1 340 Hnf1b Hoxa5 Hoxb7 Nfyb Foxo3 Klf5 Rxra Elf3 Klf1 E2f4 Klf15 320 Hoxb5 Hoxa7 Pitx2 Sox2 Trp53 Gmeb1 Gtf2b Bhlhe41 Gata3 Hdac2 Grhl1 Tal1 Elk1 E2f6 Hoxb2 Tead2 Pparg Tfdp1 Cebpz Dbp Tead3Nr1h3 Snai3 300 Sox11Pbx3 Tead4 E2f7 Sin3a Tcf7l2 Eomes Rest Rarg Taf7 E2f1 Nfya Gabpa Tbx2 Hoxc10 Foxp2 Sox10 Creb3 Sox17 E2f2 Tcf12 STRING link “Combined score” 280 Klf16 Gata2 Hcfc1 Setdb1 Atf3 Cebpa Mxi1Cebpe Taf1 Nr2f2 Gli1 Mitf Jun Elk3 Gfi1 Patz1 Nfatc4 Hoxc6 Nfe2 Fos Fosl1 Ets2 Stat5a Nr2c2 Bdp1 CSI (Connection Specificity Index) Gata6 Mafb Arid3aStat5b Yy1 Nfic Tfe3 Nr3c1 Ctcf Sp1 Creb3l1 Xbp1 Mef2a Srf Irf7 Ikzf1 Msx1 Nfil3 Pml Elf2 Mafg Usf2 Ep300 Gli2 Cebpd Fli1 Tcf7 Sp3 Gmeb2 Foxc1 Maff Fosl2 Rara Egr1 Myc Bach1 Elf4 Cebpb Lef1 Elk4 Kdm5a Crem Stat6 Irf8 Usf1 Nfia Egr2 Mafk Etv3 Hsf1 Atf7 Fosb Junb Irf1 Ets1 Atf4 Sp4 Irf3 Rela Irf5 Elf1 Irf9 Runx3 Pou2af1 Nfatc2 Jund Stat3 Bcl3 Relb Mecp2 Nfat5 Tgif1 Foxo1 Irf2 Rxrb Nfkb2 Rel Batf Spib Mef2d Essential Ddit3 Pknox1 Foxk1 Stat1 Foxp1 Non-Essential

Total network nodes (regulons): 174 Network nodes in OGEE: 154 (109 essential, 45 non essential) Network nodes not in OGEE: 20 Supplementary Figure 18

A. Pou2af1 granulocyte basophil RAS myeloid cell MCA granulocytopoietic cell 0.4 immature T cell natural killer cell classical monocyte blood cell 23 non-classical monocyte 0.0 monocyte T cell mesenchymal stem cell bladder urothelial cell hematopoietic precursor cell mesangial cell 3 macrophage 8 alveolar macrophage endocardial cell type II pneumocyte endothelial cell 24 kidney collecting duct epithelial cell cardiac muscle cell hepatocyte kidney proximal straight tubule epithelial cell fibroblast 21 bladder cell 21 stromal cell TM-SS2 basal cell luminal epithelial cell of mammary gland skeletal muscle satellite cell 12 neuroendocrine cell MCA TM-10x TM-SS2 mesenchymal cell leukocyte TM-10x late pro-B cell early pro-B cell immature B cell B cell

B. Eomes

MCA natural killer cell RAS T cell basophil 0.4 luminal epithelial cell of mammary gland myeloid cell 241 classical monocyte blood cell 0.0 kidney collecting duct epithelial cell type II pneumocyte neuroendocrine cell immature T cell cardiac muscle cell leukocyte macrophage non-classical monocyte 35 hematopoietic precursor cell granulocyte monocyte skeletal muscle satellite cell 5 bladder urothelial cell 11 mesangial cell alveolar macrophage basal cell early pro-B cell granulocytopoietic cell 59 26 231 B cell TM-SS2 stromal cell mesenchymal cell fibroblast mesenchymal stem cell MCA TM-10x TM-SS2 bladder cell immature B cell endothelial cell TM-10x late pro-B cell kidney proximal straight tubule epithelial cell hepatocyte endocardial cell

C. Tcf7

MCA RAS natural killer cell macrophage 0.6 myeloid cell 2 late pro-B cell early pro-B cell non-classical monocyte 0.0 monocyte 1 skeletal muscle satellite cell classical monocyte B cell 1 immature B cell cardiac muscle cell blood cell 7 basophil neuroendocrine cell bladder urothelial cell endothelial cell basal cell luminal epithelial cell of mammary gland 4 leukocyte 3 fibroblast granulocytopoietic cell 6 mesangial cell TM-SS2 hematopoietic precursor cell kidney collecting duct epithelial cell mesenchymal stem cell hepatocyte endocardial cell granulocyte TM-10x mesenchymal cell MCA TM-10x TM-SS2 alveolar macrophage kidney proximal straight tubule epithelial cell bladder cell type II pneumocyte stromal cell T cell immature T cell Supplementary Figure 19

A. Hsf1 bladder urothelial cell RAS mesenchymal cell 0.075 mesangial cell blood cell stromal cell myeloid cell 0.0 B cell skeletal muscle satellite cell T cell natural killer cell hepatocyte early pro-B cell kidney proximal straight tubule epithelial cell late pro-B cell endothelial cell fibroblast basal cell 11 11 15 neuroendocrine cell classical monocyte immature T cell monocyte mesenchymal stem cell endocardial cell bladder cell type II pneumocyte TM-10x TM-SS2 MCA alveolar macrophage basophil granulocytopoietic cell macrophage granulocyte kidney collecting duct epithelial cell hematopoietic precursor cell MCA TM-10x TM-SS2 non-classical monocyte leukocyte cardiac muscle cell immature B cell luminal epithelial cell of mammary gland

B. Etv3

RAS basal cell TM-10x neuroendocrine cell 0.3 mesenchymal stem cell endocardial cell mesangial cell hematopoietic precursor cell 0.0 immature T cell late pro-B cell cardiac muscle cell hepatocyte 250 fibroblast mesenchymal cell kidney proximal straight tubule epithelial cell stromal cell bladder cell skeletal muscle satellite cell kidney collecting duct epithelial cell 37 luminal epithelial cell of mammary gland bladder urothelial cell 44 type II pneumocyte T cell 47 endothelial cell granulocytopoietic cell early pro-B cell TM-SS2 granulocyte basophil 257 119 immature B cell B cell macrophage leukocyte natural killer cell MCA TM-10x TM-SS2 classical monocyte MCA monocyte non-classical monocyte alveolar macrophage

myeloid cell C. Mafb blood cell

RAS non-classical monocyte 0.6 alveolar macrophage macrophage MCA leukocyte monocyte 0.0 mesenchymal stem cell basophil stromal cell 199 endocardial cell granulocyte type II pneumocyte hepatocyte endothelial cell mesangial cell fibroblast bladder cell 60 cardiac muscle cell immature B cell B cell 1 T cell TM-10x late pro-B cell TM-SS2 1 early pro-B cell 17 immature T cell hematopoietic precursor cell mesenchymal cell 608 granulocytopoietic cell luminal epithelial cell of mammary gland skeletal muscle satellite cell basal cell bladder urothelial cell kidney proximal straight tubule epithelial cell MCA TM-10x TM-SS2 natural killer cell neuroendocrine cell kidney collecting duct epithelial cell blood cell myeloid cell classical monocyte Supplementary Figure 20

A. Irf5 monocyte RAS alveolar macrophage leukocyte MCA 0.16 myeloid cell macrophage classical monocyte non-classical monocyte 0.0 blood cell mesenchymal cell 374 stromal cell immature T cell mesenchymal stem cell kidney collecting duct epithelial cell cardiac muscle cell basal cell endocardial cell hepatocyte 169 147 bladder cell skeletal muscle satellite cell kidney proximal straight tubule epithelial cell luminal epithelial cell of mammary gland 473 mesangial cell bladder urothelial cell TM-SS2 neuroendocrine cell fibroblast endothelial cell 222 late pro-B cell 226 type II pneumocyte early pro-B cell granulocytopoietic cell 97 T cell hematopoietic precursor cell MCA TM-10x TM-SS2 immature B cell TM-10x B cell basophil granulocyte natural killer cell

B. Irf9

endothelial cell MCA RAS mesenchymal stem cell basophil 0.125 mesangial cell hematopoietic precursor cell granulocytopoietic cell stromal cell 649 0.0 early pro-B cell type II pneumocyte immature T cell neuroendocrine cell hepatocyte bladder urothelial cell luminal epithelial cell of mammary gland 160 cardiac muscle cell 159 kidney proximal straight tubule epithelial cell kidney collecting duct epithelial cell 175 late pro-B cell basal cell mesenchymal cell skeletal muscle satellite cell fibroblast TM-SS2 bladder cell 319 102 endocardial cell 520 alveolar macrophage T cell granulocyte classical monocyte myeloid cell blood cell TM-10x B cell MCA TM-10x TM-SS2 natural killer cell immature B cell leukocyte monocyte macrophage non-classical monocyte

C. Foxp1

MCA mesangial cell leukocyte RAS endocardial cell 0.5 type II pneumocyte neuroendocrine cell monocyte 5 4 non-classical monocyte 0.0 endothelial cell macrophage T cell basal cell 4 immature T cell mesenchymal cell basophil myeloid cell TM-SS2 classical monocyte 2 bladder cell 35 fibroblast natural killer cell granulocytopoietic cell hepatocyte stromal cell granulocyte kidney collecting duct epithelial cell TM-10x kidney proximal straight tubule epithelial cell cardiac muscle cell skeletal muscle satellite cell hematopoietic precursor cell blood cell luminal epithelial cell of mammary gland alveolar macrophage MCA TM-10x TM-SS2 late pro-B cell bladder urothelial cell mesenchymal stem cell early pro-B cell immature B cell B cell Supplementary Figure 21 A. Comparing GRN inference methods B. Regulon composition similarity

Irf7 Elf4 Elk3 Twist2 Gata2 Ets1 Rarg Fosl1 Hoxb5 Irf5 Crem Nfatc4 Fos Hoxb2 Irf1 Relb Hdac2 Mitf Hoxa5 Taf1 Nfkb2 Hoxa7 Rxra Pitx2 Pml Nr3c1 Sox17 Ets2 Nfia Usf1 Bcl3 Ikzf1 Elf2 Hnf1b Fli1 Kdm5a Foxp2 Xbp1 Stat4 Sin3a Runx3 Prrx2 Fosl2 Spib Jun Klf4 Sp100 Myc Klf9 Atf3 E2f2 Tead3 Cebpd Mafk Nr1h3 Irf2 Nfic Klf10 Jund Foxp4 Klf7 Pou2af1 Setdb1 Creb3 Irf8 Hcfc1 Ep300 Trp53 Fosb Gfi1 Junb Gli3 Zbtb33 Atf4 Gabpa E2f4 Vezf1 Irf9 Tef Sox10 Gtf2b Rela Yy1 Maff Bptf Nr2c2 Egr2 Stat1 Gli2 Cebpz Egr1 Creb3l1 Grhl1 Elf3 Bdp1 Eomes Pitx1 Sox9 Nr2f1 Tfe3 Sp4 Meis2 Klf15 Tcf12 Srebf1 Msx1 Ehf Maff Stat3 Tbx2 Gli1 Nfatc2 Pbx3 Klf15 Ep300 Foxc1 Atf4 Klf16 Ctcf Irf3 Ehf Foxc1 Fosb Grhl1 Elf3 Ddit3 Gli1 Rela Irf9 Elk1 Nr2f6 Creb3 Tead2 Eomes Sox11 Klf16 Ppard Mafk Tead2 Stat3 Stat1 Jund Nr2f6 E2f4 Hnf1b Srf Creb3l1 Vezf1 Ppard Spib Atf7 Ikzf1 Tead3 Junb Pitx1 Elk1 Msx1 Nr1d1 Irf8 Klf9 Egr1 Srf Tef Hcfc1 Rfxank Atf3 Atf7 Nfic Tbx2 Hdac1 Gabpa Tcf12 Hoxb5 Bdp1 Brf1 Klf7 Hsf1 Sox17 Fosl2 Nr6a1 Klf10 Pou2af1 Pitx2 Nfatc2 Rxrb Irf2 Hlf Nfatc3 E2f2 Sox2 Nr2c2 Rarg Tcf3 Fli1 Patz1 Klf4 Foxp2 Hoxa7 Xbp1 Rfx1 Ets2 Nfatc4 Hdac2 Srebf1 Egr2 Runx3 Nfia Ctcf Kdm5a Hoxb2 Myc Sp1 Tbx3 Jun Pura Pml Tfcp2 Nfat5 Bcl3 Prrx2 Nr1h3 Twist2 Mecp2 Nfkb2 Hoxa5 Cebpd Meis2 Nr2f2 Taf1 Gli3 Mitf Nr2f1 Relb Sp100 Gata6 Crem Gli2 Fosl1 Stat4 Ddit3 Ets1 Gata2 0.0 0.5 1.0 Elk3 Fraction of total genes in regulon (%) 0.0 0.04 0.08 0.12 0.16

This study (SCENIC: GRNBoost & RCisTarget) Jaccard index Ioconno et al 2015 Intersection Supplementary Figure 22

WT A. scRNA-seq (Expression)

HSCP-1 HSCP-2 Multi-Lin Monocytes

UMAP2 Granulocytes Irf8-/- Irf8-/- Other cell types

UMAP1

WT B. scRNA-seq (Regulon)

HSCP-1 HSCP-2 Multi-Lin Monocytes Granulocytes -/- UMAP2 Irf8 Other cell types Irf8-/-

UMAP1

C. Irf8 (Expression) D. Irf8 (Regulon activity)

15.0 0.25

10.0 0.20

0.15 5.0 Irf8 (RAS) 0.10 0.0 Irf8 (scaled expression) -2.5 0.05

HSCP-1 HSCP-2 Multi-Lin Monocytes Irf8-/- Granulocytes HSCP-1 HSCP-2 Multi-Lin Monocytes Irf8-/- Granulocytes

E. Irf8 regulon composition

501 400

200 116 52 29 11 2 1

intersection size 0 Set size 66 Irf8 -/-

148 Granulocytes

542 Monocytes 500