Additional file 1 of Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity
Creators: name: Zhou, B.
name: Stueve, T. R.
name: Mihalakakos, E. A.
name: Miao, L.
name: Mullen, D.
name: Wang, Y.
name: Liu, Y.
name: Luo, J.
name: Tran, E.
name: Siegmund, K. D.
name: Lynch, S. K.
name: Ryan, A. L.
name: Offringa, I. A.
name: Borok, Z.
name: Marconett, C. N.
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Fig. S1. Quality control for alveolar epithelial cell (AEC) differentiation. A) Western blots examining AT2 and AT1 cell markers during differentiation. LAMIN A/C and ACTB are the loading controls. B) Transepithelial resistance as measured in kΩ-cm2 over the course of differentiation. Error bars represent technical duplicates for each plating. C) Representative image of the cytospin staining of AT2 cell specific (TTF1, left panels) and contaminating cell markers (CD45, middle panels; Vimentin, right panels) in freshly isolated AT2 cell preparations from the indicated donors. At least 5 fields were randomly selected for counting. Red = Propidium Iodide, Green = indicated antibody. Fig. S2. Concordance of 2D AEC differentiation model with single cell RNAseq on primary lung tissue from multiple consortia. A) Single cell RNAseq analysis derived from control patients listed in IPF Cell Atlas (left) [118]. Cells were filtered based on expression of epithelial markers, specifically clusters containing EPCAM, then clustered using Seurat in R. UMAP projections are displayed. Colors indicate cluster identity. UMAP projections from IPF Cell Atlas control epithelial cells (right). Blue = cells with high expression of the indicated marker. Grey = cells lacking expression of the indicated marker. B) Differential expression of AT2 and AT1 enriched gene expression in IPF Cell Atlas plotted by -log10 FDR-corrected significance (left), concordance with differentially expressed genes in the 2D AEC differentiation model (middle). Blue = AT1 enriched genes in IPF Cell Atlas, red = AT2 enriched genes in IPF Cell Atlas. AT1 and AT2-enriched genes from the IPF cell atlas were then subset from the 2D AEC differentiation model RNAseq and plotted as a heatmap (right). Blue = little to no expression, red = high expression. C) Same analysis as for (B) was used on the Molecular Cell Atlas of Human Lung [119]. D) Same analysis was used as for (B) on control mice from lung single cell analysis [26]. Fig. S3. FAIRE-seq quality assessment. A) IGV image of FAIRE-seq data. FAIRE was performed on AT2 (D0), AT-transitional phenotype (D4), and AT1-like (D6) cells for Donor 1. Region surrounding FOXA2 locus, which is expressed in both AT2 and AT1-like cells, is shown. FAIRE-seq BigWig tracks are displayed with called FAIRE peaks directly below. B) Table of mapping statistics for FAIRE-seq data. C) Peak saturation plot for FAIRE-seq data. Inset = overlap between regions called FAIRE peaks in AT2 (D0), AT1.5 (D4) and AT1-like cells (D6). Fig. S4. H3K4me1 ChIP-seq quality assessment. A) Table of H3K4me1 mapping statistics. B) Peak saturation plot. C) Tag distribution of ChIP-seq read densities at FAIRE peak locations common to both AT2 (D0) and AT1-like (D6) cells. D) Diffbind correlation plot between samples. Condition = Timepoint during differentiation. Black = AT2 (D0), red = AT1.5 (D4), blue = AT1-like (D6). E) Overlap in called peak locations between biological replicates. Fig. S5. H3K27Ac ChIP-seq quality assessment. A) Table of H3K27Ac mapping statistics. B) Peak saturation plot. C) Tag distribution of ChIP-seq read densities at FAIRE peak locations common to both AT2 (D0) and AT1-like (D6) cells. D) Diffbind correlation plot between samples. Condition = Timepoint during differentiation. Black = AT2 (D0), red = AT1.5 (D4), blue = AT1-like (D6). E) Overlap in called peak locations between biological replicates. Fig. S6. Transcription factor binding site enrichment in separate subsets of A549 enhancers. A) Bar plot of top 25 transcription factor binding site predicted motifs within A549 enhancers subset by overlap with either AT1 enhancers (blue), AT2 enhancers (orange), or only present in A549 (grey). AT1 vs. AT2 enhancer motif correlation = 0.975, p