1. Volume 20, Issue 3, Pages 709-720 (July 2017)
Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq 
Aurelie Quillien, Mary Abdalla, Jun Yu, Jianhong Ou, Lihua Julie Zhu, Nathan D. Lawson 
Cell Reports 
Volume 20, Issue 3, Pages (July 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 20, 709-720DOI: (10.1016/j.celrep.2017.06.070)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 ATAC-Seq Data from Isolated Zebrafish Nuclei
(A) Schematic for isolation of nuclei from Tg(fli1a:egfp)y1 zebrafish embryos.
(B) Heatmaps showing density of mapped ATAC-seq reads from single biological replicates 1 kb up and downstream of ENSEMBL-annotated transcriptional start sites (TSS) in Zv9. Separate heatmaps for fragments less than 100 bp and between 180 and 247 bp are shown.
(C and D) Density plots flanking Zv9 TSS from (C) GFP-negative and (D) GFP-positive nuclei for mapped reads shown in (B).
(E–H) Heatmaps showing localization of ATAC-seq reads with (E) H3K4me3, (F) H3K4me1, (G) H3K27ac, or (H) zCNE. Intervals flanking indicated feature are shown in kilobases.
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4. Figure 2 Mapping of ATAC-Seq Reads at Known Endothelial Enhancers
(A–C) Visualization of ATAC-seq from a single biological replicate, whole-embryo ChIP-seq mapped reads for H3K4me1, H3K4me3, and H3K27ac, and conservation from the UCSC Genome Browser.
(A) etv2 locus. Red box is known active endothelial enhancer.
(B) fli1a locus. Red box indicates known active endothelial enhancer.
(C) nrarpa locus. Black boxes are open regions in both GFP-positive and -negative nuclei.
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5. Figure 3
Identification and Characterization of Cell-Type-Specific Open Chromatin
(A) Heatmap of log2 fold change in GFP+/GFP– ATAC-seq read density (FDR ≤ 0.05; biological triplicates).
(B–D) Density plots of mapped reads from H3K4me1, H3K4me3, and H3K27ac whole-embryo ChIP-seq against ATAC-seq elements specific to (B) GFP-positive, (C) GFP-negative, or (D) common elements.
(E) Proportion of elements of indicated class that associate with TSS or H3K4me3.
(F) Proportion of elements concordant with H3K27ac or zCNEs.
(G and H) Expression level (by RNA-seq) in log2 fragments per kilobase per million reads (FPKM) in GFP-positive or GFP-negative cells from Tg(fli1a:egfp)y1 for genes with TSS open specifically in (G) GFP-positive or (H) GFP-negative nuclei; ∗∗∗∗p < Error bars represent ± SEM.
(I–K) Plots comparing log2 fold change in expression (by RNA-seq), between GFP-positive and -negative cells from Tg(fli1a:egfp)y1 embryos to number of flanking (I) GFP-positive, (J) GFP-negative, or (K) common elements. Only genes with GFP+/GFP– log2 fold change ≥1, or ≤ −1 (FDR ≤ 0.05) were included.
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6. Figure 4 GFP-Positive Elements Can Drive Endothelial Gene Expression
(A) Average read depth, in counts per million (cpm), at each element used for reporter assays. Data are represented as mean ± SD from triplicate ATAC-seq libraries. All elements display GFP+/GFP– log2 fold change >1 (p < ; FDR < 0.05; biological triplicates). Name of adjacent endothelial gene shown on x axis with distance (in kilobases) and direction (“+” = downstream; “–” = upstream) of enhancer relative to TSS.
(B) Tol2 plasmid backbone used for reporter assays.
(C, E, G, and I) Overlays of green and red fluorescent images from embryos injected with reporter constructs. Lateral views: dorsal is up, and anterior is to the left. Ratios in left bottom denote number of embryos with GFP expression over number of cryaa:mcherry-expressing embryos from replicate injections. Embryos injected with (C) reporter with only basal promoter driving EGFP, or reporter with elements (E) downstream of mafbb, (G) upstream of nrp1b or (I) tmem88a. (D, F, and H) Mapped reads flanking indicated genes from GFP-positive and -negative nuclei isolated from Tg(fli1a:egfp)y1 embryos.
(E, G, and I) White arrowheads denote low-level expression in trunk endothelial cells. Black boxes are elements used in reporter assays. Scale bar, 250 μm.
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7. Figure 5
Pairing Cognate Promoters and Enhancers Improves Endothelial Expression
(A, E, and I) GFP-positive (green) and GFP-negative (black) ATAC-seq read density in nuclei at (A) lmo2, (E) clec14a, and (I) dll4 loci. Black boxes are putative enhancer elements; gray boxes denote region used as a promoter.
(B–D, F–H, and J–L) Overlays of green and red fluorescence from embryos injected with enhancer reporter constructs. Ratios in left bottom are number of embryos with endothelial GFP over the total number of cryaa:mcherry-expressing embryos from replicate injections. Lateral views, dorsal is up, anterior to the left. Embryos injected with reporter construct containing (B, F, J) gene-specific promoter, (C, G, K) enhancer and basal promoter, or (D, H, L) enhancer and cognate promoter for indicated genes upstream of EGFP. Scale bar, 250 μm.
Cell Reports  , DOI: ( /j.celrep )
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