ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing

Journal name:
Nature Methods
Volume:
13,
Pages:
1013–1020
Year published:
DOI:
doi:10.1038/nmeth.4031
Received
Accepted
Published online

Abstract

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

At a glance

Figures

  1. ATAC-see visualizes the accessible genome in situ.
    Figure 1: ATAC-see visualizes the accessible genome in situ.

    (a) Schematic of ATAC-see. (b) Genome browser tracks of ATAC-seq libraries from GM12878 cells (GM) generated by using different Tn5 transposases: green, Nextera Tn5; blue, Atto Tn5; orange, previously published data3. A 50-kb scale bar and genome locations are indicated at the top of the tracks; gene names (HAUS5, etc.) are shown at the bottom. Chr, chromosome; hg19, human genome. (c) Left panel, genome-wide comparison of ATAC-seq reads of GM12878 libraries (GM) prepared by using either Nextera Tn5 or Atto Tn5. Right panel, TSS enrichment of GM12878 ATAC-seq libraries transposed with different Tn5. Green, Nextera Tn5; blue, Atto Tn5; orange, previously published data3. (d) Genome-wide comparison of ATAC-seq reads of HT1080 library prepared with or without fixation. Left, scatter plot of all data points; right, metagene analysis centered on transcriptional start sites (TSS).

  2. ATAC-see enables imaging and sequencing of the accessible genome in the same cells.
    Figure 2: ATAC-see enables imaging and sequencing of the accessible genome in the same cells.

    (a) Representative image of ATAC-see result in HT1080 cells. Very limited ATAC-see signal is observed in +EDTA negative control (quantified in Supplementary Fig. 3a). Merge, merged image of DAPI and ATAC-see. Scale bars, 2 μm. (b) Multimodal imaging combining ATAC-see with immunofluorescence. The representative images employ costaining with nucleus lamin B1 and/or mitochondrial protein marker (Mito) to show ATAC-see signals overlapping with mitochondria outside of the nucleus. Scale bar, 2 μm. (c) Genomic tracks of ATAC-seq data from standard protocol and Atto-Tn5 on slide after imaging. x-axis, genomic coordinates; y-axis, normalized ATAC-seq read counts. 50-kb scale bar and genome locations are indicated at the top; gene names (Myc, etc.) are shown at the bottom. Chr, chromosome; hg19, human genome. (d) Genome-wide comparisons of standard ATAC-seq data with Atto Tn5 on-slide data after imaging. Left, scatter plot of all data points; right, metagene analysis centered on transcriptional start sites (TSS).

  3. Cell-type-specific accessible-chromatin organization in the intact nucleus.
    Figure 3: Cell-type-specific accessible-chromatin organization in the intact nucleus.

    (a) Image analysis. For each cell type (organized in rows), we display four columns (i–iv, left to right): (i) a representative ATAC-see image (red, ATAC-see; blue, DAPI; scale bar, 2 μm); (ii) signal intensity of ATAC-see and DAPI as a function of distance from nuclear periphery. Each trace is one nucleus, and n = number of nuclei analyzed; (iii) correlation of ATAC-see and DAPI signal intensity. Pearson correlation (r) is indicated; (iv) ATAC-see clusters, quantified as the ratio of ATAC-see bright areas versus the total nucleus area. (b) Unique feature of ATAC-seq from human neutrophil (Neuts) after imaging. Left, genome browser track of ATAC-seq in human neutrophil, H3K27Ac layer from ENCODE 7 cell lines, and lamin-associated domains (LADs) published by the Netherlands Cancer Institute (NKI). x-axis, genomic coordinates; y-axis, ATAC-seq-normalized read counts. The gray line indicates the location of bacterial artifical chromosome (BAC) chosen for DNA FISH in c. Right, metagene plot of human neutrophil ATAC-seq signal centered on the boundary between NKI LADs and neighboring sequences. The dashed black line indicates the boundary of LADs, and the thick black line on the top of the graph presents the distance to LADs boundary. (c) Left, example of DNA FISH from indicated BAC (in b) in human neutrophil; the white arrow indicates the FISH signal (red) at the neutrophil periphery. Right, location quantification of DNA FISH signal (percentage in y-axis) from the indicated BACs (x-axis) in the human neutriophil. The nucleus periphery was defined as a distance between DNA FISH signal to DAPI staining edge <0.1 μm (see Online Methods). n, number of alleles counted in DNA FISH. P values were calculated by a binomial test.

  4. ATAC-see and ATAC-seq reveal the dynamic chromatin organization during human NETosis.
    Figure 4: ATAC-see and ATAC-seq reveal the dynamic chromatin organization during human NETosis.

    DAPI, stain used to stain the nucleus; merge, merged image of DAPI and ATAC-see. (a) Representative ATAC-see images in the indicated conditions. PMA stimulation timecourse, PAD4 inhibitor treatment (PAD4i). Scale bars, 2 μm. (b) Epigenomic landscape of NETosis. Left column, genomic tracks of ATAC-seq data from the indicated conditions. Locations of NKI lamin-associated domains (LADs) are indicated. The x-axis represents genomic coordinates; the y-axis represents ATAC-seq normalized read counts. Middle column, metagene plot of ATAC-seq signal centered on the boundary between NKI LADs and neighboring sequences. The top plot is the same as the right panel in Figure 3b and is reproduced here for clarity as the baseline in this timecourse. Right column, ATAC-seq insert size distribution for the corresponding samples. Diagnostic insert sizes for accessible DNA, mononucleosome, and dinucleosome are labeled. (c) Proposed model of NETosis illustrates the coordinated dynamics of nuclear architecture, accessible genome reorganization, and genome disassembly.

  5. ATAC-see reveals cell-cycle-specific genome accessibility.
    Figure 5: ATAC-see reveals cell-cycle-specific genome accessibility.

    (a) Flow cytometry with ATAC-see. Dot plot of signal intensity in dual staining for DAPI and ATAC-see of GM12878 cells; results showed four groups of cells: G1 low, G1 high, S phase, and G2. (b) Quantitation of DAPI (left) and ATAC-see (right) signals from different groups. (c) Cyclin E1 staining in ATAC-see-sorted G1-high and G1-low cells. Left panel, representative images from confocal microscopy. Scale bars, 2 μm. Right panel is a box plot depicting signal intensity measurement. n, cell number; ***, P < 0.001, Student t-test. (d) Heatmap shows cluster of different ATAC-seq accessible regions between the G1-high and G1-low cells (FD > 2, FDR < 0.05); each group has one replicate. (e) The volcano plot represents genome-wide comparisons of accessible regions in G1-high versus G1-low cells. (f) The density histograms represent the distribution of the more accessible regions in G1-high and G1-low cells across the transcription starting sites (TSS). The more accessible regions in the two groups were color coded.

  6. Validation of ATAC-seq library from Atto-Tn5
    Supplementary Fig. 1: Validation of ATAC-seq library from Atto-Tn5

    a, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 in GM12878 cells (GM). b, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in GM12878 cells (GM). c, Insert size distribution of GM12878 ATAC-seq libraries transposed by Nextera Tn5. d, Insert size distribution of GM12878 cells ATAC-seq libraries from Atto-Tn5. e, Calculation of regulatory element enrichment from ChromHMM in previously published GM12878 ATAC-seq libraries and new ATAC-seq libraries using either Atto-Tn5 or Illumina Nextera Tn5. f: density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in GM12878 from Atto-Tn5 and Nextera Tn5 among technical replicates and different enzyme.

  7. Validation of ATAC-seq library in fixed cells
    Supplementary Fig. 2: Validation of ATAC-seq library in fixed cells

    a, Insert size distribution of HT1080 cells ATAC-seq with standard protocol. b, Insert size distribution of fixed HT1080 cells ATAC-seq without reverse crosslinking step. c, Genomic tracks of ATAC-seq data from HT1080 cells among different conditions: No fixation, with fixation. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. d, Insert size distribution of fixed HT1080 cells ATAC-seq with reverse crosslinking step. e, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in non-fixed HT1080 cells (HT). f, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in fixed HT1080 cells (HT) with reverse crosslinking.

  8. Confirmation of ATAC-see principle
    Supplementary Fig. 3: Confirmation of ATAC-see principle

    a, Left: representative whole frame images of ATAC-see from normal reaction and with 50 mM EDTA treated in HT1080 cells. The white squares in the DAPI channel indicate the cropped cells in Figure 2a. Scale bar=2 μm. Right: Signal intensity of ATAC-see was quantified in both normal reaction and 50 mM EDTA control samples. 20 cells were counted in each condition with independent replication, ** (p<0.005, student t-test), error bar is standard deviation. b, Representative whole frame images of ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in HT1080 cells. The white square in the DAPI channel indicates the cropped cells in Figure 2b. Scale bar=2 μm. c, Correlation coefficient of ATAC-see with different epigenetic markers and active form of RNAP II in HT1080 cells (n= cell number); RNAPII Ser-2 P = RNA polymerase II phosphorylation ser-2, and RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. d, Representative images of ATAC-see co-staining with different epigenetic markers and active form of RNAP II in HT1080 cells. e, ATAC-see and XIST RNA-FISH in mouse Neural Progenitor Cells. Upper panel: representative images (the white arrows in the ATAC-see panel indicate the location of XIST RNA FISH signal); lower panel: signal intensity quantification of ATAC-see within and outside of XIST area. 30 cells were counted in each condition with independent replicate, ** (p<0.005, student t-test), error bar is standard deviation.

  9. Validation of ATAC-seq library after ATAC-see imaging
    Supplementary Fig. 4: Validation of ATAC-seq library after ATAC-see imaging

    a, Schematic workflow of ATAC-seq library preparation after ATAC-see imaging. b, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 on slides from HT1080 cells (HT). c, density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in HT1080(HT) from Atto-Tn5 (on slide) and Nextera Tn5 (in solution) among technical replicates and different enzyme. d, Sensitivity assay (with different input) of global DNA accessibility of ATAC-seq after imaging (on slides) from HT1080 cells.

  10. ATAC-see in different cell types
    Supplementary Fig. 5: ATAC-see in different cell types

    a, Representative whole frame images of ATAC-see from different cell types. Scale bar=2 μm. Multi images (n>5) were taken in each cell type with independent replicates. b, Cell type specific accessible chromatin organization in the intact nucleus. The violin plot represents of the correlation coefficients between ATAC-see signal and DAPI signal per nucleus in different cell types. Each cell type has a unique profile, and neutrophils stand out as an outlier. B-cell = B-lymphoblastoid GM12878 cells.

  11. Systematic imaging processing of ATAC-see
    Supplementary Fig. 6: Systematic imaging processing of ATAC-see

    a, Schematic of image processing workflow of making mitochondria masks from ATAC-see and define the bright area of ATAC-see signal in the nucleus. b, Analysis of two different ATAC-see patterns in CD4+ T cells. The two plots in the upper panel represent the radial distribution of ATAC-see signal intensity and DAPI signal intensity in two different groups of CD4+ T cells. The blue line shows the ATAC-see signal intensity is low at the nucleus periphery, the green line illustrates the ATAC-see signal form a rim structure at the nucleus periphery (“Cap pattern”) and the red line indicates the means of all signal intensity. The line plot in the middle panel shows the ratio of ATAC-see signal intensity and DAPI signal intensity. The scatter plot in the lower panel represents the correlation of ATAC-see signal intensity and DAPI signal intensity among the population cells. The red lines in the plot represent the average values of single intensity from all groups.

  12. Cell type specific accessible chromatin organization in the intact nucleus.
    Supplementary Fig. 7: Cell type specific accessible chromatin organization in the intact nucleus.

    For each cell type (organized in rows), we display from left to right columns: (i) A representative ATAC-see image (red color is ATAC-see and blue is DAPI, scale bar = 2 μm). (ii) Signal intensity of ATAC-see and DAPI as a function of distance from nuclear periphery. Each trace is one nucleus; n= number of nuclei analyzed. (iii) Correlation of ATAC-see and DAPI signal intensity; Pearson correlation (r) is indicated. (iv) ATAC-see clusters, quantified as the ratio of ATAC-see bright areas vs total nucleus area.

  13. Unique pattern of ATAC-see in the human neutrophil
    Supplementary Fig. 8: Unique pattern of ATAC-see in the human neutrophil

    a, Representative whole frame images show ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in human neutrophils. The dotted lines in the Lamin B1 panel show the location of the nucleus periphery based on DAPI staining. Multi images (n>5) were taken with independent replicates. Scale bar=2 μm. b, The similarity of ATAC-seq libraries after imaging (on slide) with ATTO-590 from different donors. c, The boxplot represents the ATAC-seq peaks enrichment within NKI LADs and outside of NKI LADs, 0= outside of LADs, 1= within LADs. d, the black squares show the location of BAC clones chosen for DNA FISH according to the genomic tracks of ATAC-seq data in human neutrophil; BAC clones were chosen from both LAD region (RP11-626N18, RP11-832P24) and none LAD regions (RP11-63J14, RP11-637D5, RP11-368K11, RP11-116A9). e, Representative DNA FISH image in human neutrophils: the left panel: extended focus, right four panels: side view of 3D images. X-Y= X dimension and Y dimension, X-Z= X dimension and Z dimension. Scale bar=2 μm.

  14. Immunostaining in human neutrophils and NETosis
    Supplementary Fig. 9: Immunostaining in human neutrophils and NETosis

    a, Immunostaining of epigenetic markers in human neutrophil. RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. Multi images (n>5) were taken with independent replicates in each staining. b, The representative images show DAPI staining of control, PMA and PAD4 inhibitor (PAD4i) treated human neutrophils. Scale bar=2 μm. c, The bar graph presents the quantification of NETosis in PMA and PAD4 inhibitor treated human neutrophils based on DAPI staining, Error bar= standard deviation; n=60x2 for each condition. d, Representative whole frame images of H3 citrullination staining in control, 5 h PMA treated and 5 h PAD4 inhibitor (PAD4i) treated human neutrophils. Multi images (n=5x2) were taken in each condition. Scale bar=2 μm.

  15. ATAC-see and -seq in the human NETosis.
    Supplementary Fig. 10: ATAC-see and –seq in the human NETosis.

    a, Representative images ATAC-see in 3h PMA stimulation human neutrophils. Scale bar=2 μm.

    b, Epigenomic landscape of 3h PMA stimulation human neutrophils. Left column: Genomic tracks of ATAC-seq data. Locations of NKI Lamin associated domains (LADs) are indicated. X-axis is genomic coordinates; Y-axis is ATAC-seq normalized read counts. Middle: Metagene plot of ATAC-seq signal centered on the boundary between NKI LADs and neighboring sequences. Right: ATAC-seq insert size distribution for the corresponding samples. Diagnostic insert sizes for accessible DNA, mononucleosome, and di-nucleosome are labeled.

  16. FACS analysis of ATAC-see and DAPI dual staining in GM12878 cells.
    Supplementary Fig. 11: FACS analysis of ATAC-see and DAPI dual staining in GM12878 cells.

    a, Left panel: cell cycle histogram from DAPI staining. Right panel: ATAC-see signal intensity histogram. b, violin plots show the ATAC-see signal intensity of the sorted cells measured by confocal microscopy. n= cell number measured under the confocal microscopy. *** (p<0.001, ANOVA test). c, Heatmap of the correlation coefficient of different FACS sorted groups. Each group contains independent replicate. d, Heatmap represents the cluster of accessible regions (FD>2, FDR<0.05) in FACS sorted groups: G1 low, G1 high, S phase and G2. Each group contains independent replicate. e, genome-wide comparison of accessible regions between different ATAC-see sorted groups: G1 low vs. G1 high; S vs. G1 high; G2 vs. S; G1 low vs. G2.

  17. Classification of accessible regions in G1
    Supplementary Fig. 12: Classification of accessible regions in G1

    a, Genomic tracks of ATAC-seq data from ATAC-see G1 low and G1 high group after FACS sorting. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. b, ChromHMM classification of accessible regions in asynchronized GM12878 cells (left), more accessible G1 high (middle) and more accessible G1 low regions (right). Each chromatin state was color-coded.

  18. FACS analysis of ATAC-see and cell surface marker staining in mouse bone marrow progenitor cells.
    Supplementary Fig. 13: FACS analysis of ATAC-see and cell surface marker staining in mouse bone marrow progenitor cells.

    a: FACS plots was gated (see Methods) as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and band neutrophils; b: Representative images of sorted populations shown in (a) by confocal microscopy, and multi images (n>5) were taken with independent replicates in each group.

Videos

  1. Supplementary Video 1
    Video 1: Supplementary Video 1
    HT1080 cells 3D movie of ATAC-see HT1080 cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  2. Supplementary Video 2
    Video 2: Supplementary Video 2
    HeLa cells 3D movie of ATAC-see HeLa cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  3. Supplementary Video 3
    Video 3: Supplementary Video 3
    CD4+ T cells 3D movie of ATAC-see CD4+ T cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  4. Supplementary Video 4
    Video 4: Supplementary Video 4
    B lymphocyte cell line GM12878 cells 3D movie of ATAC-see B lymphocyte cell line GM12878 cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  5. Supplementary Video 5
    Video 5: Supplementary Video 5
    Human neutrophils 3D movie of ATAC-see Human neutrophils 3D movie of Lamin B1 (green), mitochondria protein staining (yellow), ATAC-see (red) and DAPI staining (blue).

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Center for Personal Dynamic Regulomes, Stanford University, Stanford, California, USA.

    • Xingqi Chen,
    • Ying Shen,
    • Jason D Buenrostro,
    • Ulrike Litzenburger,
    • Seung Woo Cho,
    • Ansuman T Satpathy,
    • Ava C Carter,
    • William J Greenleaf &
    • Howard Y Chang
  2. Department of Bioengineering, Stanford University, Stanford, California, USA.

    • Will Draper,
    • Rajarshi P Ghosh &
    • Jan T Liphardt
  3. Department of Genetics, Stanford University, Stanford, California, USA.

    • Jason D Buenrostro &
    • William J Greenleaf
  4. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.

    • Alexandra East-Seletsky &
    • Jennifer A Doudna
  5. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.

    • Alexandra East-Seletsky &
    • Jennifer A Doudna
  6. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.

    • Jennifer A Doudna
  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Jennifer A Doudna
  8. Department of Applied Physics, Stanford University, Stanford, California, USA.

    • William J Greenleaf

Contributions

X.C., W.J.G., and H.Y.C. conceived and designed the study. X.C., J.D.B., U.L., A.T.S., A.C.C., and R.P.G. performed experiments. Y.S. and X.C. performed genomic data analysis. W.D., X.C. and J.T.L. conducted image analyses. S.W.C., A.E.-S., and J.A.D. generated reagents. X.C. and H.Y.C. wrote the manuscript with input from all authors. H.Y.C. supervised all aspects of this work.

Competing financial interests

H.Y.C. and W.J.G. are cofounders of Epinomics. Stanford University has filed a patent on ATAC-see, on which X.C., J.D.B., W.J.G., and H.Y.C. are coinventors.

Corresponding author

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Validation of ATAC-seq library from Atto-Tn5 (128 KB)

    a, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 in GM12878 cells (GM). b, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in GM12878 cells (GM). c, Insert size distribution of GM12878 ATAC-seq libraries transposed by Nextera Tn5. d, Insert size distribution of GM12878 cells ATAC-seq libraries from Atto-Tn5. e, Calculation of regulatory element enrichment from ChromHMM in previously published GM12878 ATAC-seq libraries and new ATAC-seq libraries using either Atto-Tn5 or Illumina Nextera Tn5. f: density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in GM12878 from Atto-Tn5 and Nextera Tn5 among technical replicates and different enzyme.

  2. Supplementary Figure 2: Validation of ATAC-seq library in fixed cells (102 KB)

    a, Insert size distribution of HT1080 cells ATAC-seq with standard protocol. b, Insert size distribution of fixed HT1080 cells ATAC-seq without reverse crosslinking step. c, Genomic tracks of ATAC-seq data from HT1080 cells among different conditions: No fixation, with fixation. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. d, Insert size distribution of fixed HT1080 cells ATAC-seq with reverse crosslinking step. e, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in non-fixed HT1080 cells (HT). f, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in fixed HT1080 cells (HT) with reverse crosslinking.

  3. Supplementary Figure 3: Confirmation of ATAC-see principle (236 KB)

    a, Left: representative whole frame images of ATAC-see from normal reaction and with 50 mM EDTA treated in HT1080 cells. The white squares in the DAPI channel indicate the cropped cells in Figure 2a. Scale bar=2 μm. Right: Signal intensity of ATAC-see was quantified in both normal reaction and 50 mM EDTA control samples. 20 cells were counted in each condition with independent replication, ** (p<0.005, student t-test), error bar is standard deviation. b, Representative whole frame images of ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in HT1080 cells. The white square in the DAPI channel indicates the cropped cells in Figure 2b. Scale bar=2 μm. c, Correlation coefficient of ATAC-see with different epigenetic markers and active form of RNAP II in HT1080 cells (n= cell number); RNAPII Ser-2 P = RNA polymerase II phosphorylation ser-2, and RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. d, Representative images of ATAC-see co-staining with different epigenetic markers and active form of RNAP II in HT1080 cells. e, ATAC-see and XIST RNA-FISH in mouse Neural Progenitor Cells. Upper panel: representative images (the white arrows in the ATAC-see panel indicate the location of XIST RNA FISH signal); lower panel: signal intensity quantification of ATAC-see within and outside of XIST area. 30 cells were counted in each condition with independent replicate, ** (p<0.005, student t-test), error bar is standard deviation.

  4. Supplementary Figure 4: Validation of ATAC-seq library after ATAC-see imaging (168 KB)

    a, Schematic workflow of ATAC-seq library preparation after ATAC-see imaging. b, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 on slides from HT1080 cells (HT). c, density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in HT1080(HT) from Atto-Tn5 (on slide) and Nextera Tn5 (in solution) among technical replicates and different enzyme. d, Sensitivity assay (with different input) of global DNA accessibility of ATAC-seq after imaging (on slides) from HT1080 cells.

  5. Supplementary Figure 5: ATAC-see in different cell types (143 KB)

    a, Representative whole frame images of ATAC-see from different cell types. Scale bar=2 μm. Multi images (n>5) were taken in each cell type with independent replicates. b, Cell type specific accessible chromatin organization in the intact nucleus. The violin plot represents of the correlation coefficients between ATAC-see signal and DAPI signal per nucleus in different cell types. Each cell type has a unique profile, and neutrophils stand out as an outlier. B-cell = B-lymphoblastoid GM12878 cells.

  6. Supplementary Figure 6: Systematic imaging processing of ATAC-see (148 KB)

    a, Schematic of image processing workflow of making mitochondria masks from ATAC-see and define the bright area of ATAC-see signal in the nucleus. b, Analysis of two different ATAC-see patterns in CD4+ T cells. The two plots in the upper panel represent the radial distribution of ATAC-see signal intensity and DAPI signal intensity in two different groups of CD4+ T cells. The blue line shows the ATAC-see signal intensity is low at the nucleus periphery, the green line illustrates the ATAC-see signal form a rim structure at the nucleus periphery (“Cap pattern”) and the red line indicates the means of all signal intensity. The line plot in the middle panel shows the ratio of ATAC-see signal intensity and DAPI signal intensity. The scatter plot in the lower panel represents the correlation of ATAC-see signal intensity and DAPI signal intensity among the population cells. The red lines in the plot represent the average values of single intensity from all groups.

  7. Supplementary Figure 7: Cell type specific accessible chromatin organization in the intact nucleus. (168 KB)

    For each cell type (organized in rows), we display from left to right columns: (i) A representative ATAC-see image (red color is ATAC-see and blue is DAPI, scale bar = 2 μm). (ii) Signal intensity of ATAC-see and DAPI as a function of distance from nuclear periphery. Each trace is one nucleus; n= number of nuclei analyzed. (iii) Correlation of ATAC-see and DAPI signal intensity; Pearson correlation (r) is indicated. (iv) ATAC-see clusters, quantified as the ratio of ATAC-see bright areas vs total nucleus area.

  8. Supplementary Figure 8: Unique pattern of ATAC-see in the human neutrophil (134 KB)

    a, Representative whole frame images show ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in human neutrophils. The dotted lines in the Lamin B1 panel show the location of the nucleus periphery based on DAPI staining. Multi images (n>5) were taken with independent replicates. Scale bar=2 μm. b, The similarity of ATAC-seq libraries after imaging (on slide) with ATTO-590 from different donors. c, The boxplot represents the ATAC-seq peaks enrichment within NKI LADs and outside of NKI LADs, 0= outside of LADs, 1= within LADs. d, the black squares show the location of BAC clones chosen for DNA FISH according to the genomic tracks of ATAC-seq data in human neutrophil; BAC clones were chosen from both LAD region (RP11-626N18, RP11-832P24) and none LAD regions (RP11-63J14, RP11-637D5, RP11-368K11, RP11-116A9). e, Representative DNA FISH image in human neutrophils: the left panel: extended focus, right four panels: side view of 3D images. X-Y= X dimension and Y dimension, X-Z= X dimension and Z dimension. Scale bar=2 μm.

  9. Supplementary Figure 9: Immunostaining in human neutrophils and NETosis (175 KB)

    a, Immunostaining of epigenetic markers in human neutrophil. RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. Multi images (n>5) were taken with independent replicates in each staining. b, The representative images show DAPI staining of control, PMA and PAD4 inhibitor (PAD4i) treated human neutrophils. Scale bar=2 μm. c, The bar graph presents the quantification of NETosis in PMA and PAD4 inhibitor treated human neutrophils based on DAPI staining, Error bar= standard deviation; n=60x2 for each condition. d, Representative whole frame images of H3 citrullination staining in control, 5 h PMA treated and 5 h PAD4 inhibitor (PAD4i) treated human neutrophils. Multi images (n=5x2) were taken in each condition. Scale bar=2 μm.

  10. Supplementary Figure 10: ATAC-see and –seq in the human NETosis. (57 KB)

    a, Representative images ATAC-see in 3h PMA stimulation human neutrophils. Scale bar=2 μm.

    b, Epigenomic landscape of 3h PMA stimulation human neutrophils. Left column: Genomic tracks of ATAC-seq data. Locations of NKI Lamin associated domains (LADs) are indicated. X-axis is genomic coordinates; Y-axis is ATAC-seq normalized read counts. Middle: Metagene plot of ATAC-seq signal centered on the boundary between NKI LADs and neighboring sequences. Right: ATAC-seq insert size distribution for the corresponding samples. Diagnostic insert sizes for accessible DNA, mononucleosome, and di-nucleosome are labeled.

  11. Supplementary Figure 11: FACS analysis of ATAC-see and DAPI dual staining in GM12878 cells. (191 KB)

    a, Left panel: cell cycle histogram from DAPI staining. Right panel: ATAC-see signal intensity histogram. b, violin plots show the ATAC-see signal intensity of the sorted cells measured by confocal microscopy. n= cell number measured under the confocal microscopy. *** (p<0.001, ANOVA test). c, Heatmap of the correlation coefficient of different FACS sorted groups. Each group contains independent replicate. d, Heatmap represents the cluster of accessible regions (FD>2, FDR<0.05) in FACS sorted groups: G1 low, G1 high, S phase and G2. Each group contains independent replicate. e, genome-wide comparison of accessible regions between different ATAC-see sorted groups: G1 low vs. G1 high; S vs. G1 high; G2 vs. S; G1 low vs. G2.

  12. Supplementary Figure 12: Classification of accessible regions in G1 (80 KB)

    a, Genomic tracks of ATAC-seq data from ATAC-see G1 low and G1 high group after FACS sorting. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. b, ChromHMM classification of accessible regions in asynchronized GM12878 cells (left), more accessible G1 high (middle) and more accessible G1 low regions (right). Each chromatin state was color-coded.

  13. Supplementary Figure 13: FACS analysis of ATAC-see and cell surface marker staining in mouse bone marrow progenitor cells. (99 KB)

    a: FACS plots was gated (see Methods) as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and band neutrophils; b: Representative images of sorted populations shown in (a) by confocal microscopy, and multi images (n>5) were taken with independent replicates in each group.

Video

  1. Video 1: Supplementary Video 1 (441 KB, Download)
    HT1080 cells 3D movie of ATAC-see HT1080 cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  2. Video 2: Supplementary Video 2 (664 KB, Download)
    HeLa cells 3D movie of ATAC-see HeLa cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  3. Video 3: Supplementary Video 3 (474 KB, Download)
    CD4+ T cells 3D movie of ATAC-see CD4+ T cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  4. Video 4: Supplementary Video 4 (584 KB, Download)
    B lymphocyte cell line GM12878 cells 3D movie of ATAC-see B lymphocyte cell line GM12878 cells 3D movie of ATAC-see (red) and DAPI staining (blue).
  5. Video 5: Supplementary Video 5 (945 KB, Download)
    Human neutrophils 3D movie of ATAC-see Human neutrophils 3D movie of Lamin B1 (green), mitochondria protein staining (yellow), ATAC-see (red) and DAPI staining (blue).

PDF files

  1. Supplementary Text and Figures (5,383 KB)

    Supplementary Figures 1–13 and Supplementary Tables 1 and 2.

Additional data