ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing (original) (raw)

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Acknowledgements

We thank S. Kim (Stanford) for FACS access. This work was supported by NIH grant P50-HG007735 (to H.Y.C. and W.J.G.), the Life Extension Foundation (to H.Y.C.), NCI Physical Sciences Oncology Center grant U54CA143836 (to J.T.L.), and National Institute of Biomedical Imaging and Bioengineering (NIBIB)/4D Nucleome Roadmap Initiative grant 1U01EB021237 (to J.T.L.).

Author information

Authors and 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

Authors

1. Xingqi Chen
2. Ying Shen
3. Will Draper
4. Jason D Buenrostro
5. Ulrike Litzenburger
6. Seung Woo Cho
7. Ansuman T Satpathy
8. Ava C Carter
9. Rajarshi P Ghosh
10. Alexandra East-Seletsky
11. Jennifer A Doudna
12. William J Greenleaf
13. Jan T Liphardt
14. Howard Y Chang

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.

Corresponding author

Correspondence toHoward Y Chang.

Ethics declarations

Competing 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.

Integrated supplementary information

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Tables 1 and 2. (PDF 5257 kb)

Supplementary Video 1

HT1080 cells 3D movie of ATAC-see HT1080 cells 3D movie of ATAC-see (red) and DAPI staining (blue). (MOV 441 kb)

Supplementary Video 2

HeLa cells 3D movie of ATAC-see HeLa cells 3D movie of ATAC-see (red) and DAPI staining (blue). (MOV 663 kb)

Supplementary Video 3

CD4+ T cells 3D movie of ATAC-see CD4+ T cells 3D movie of ATAC-see (red) and DAPI staining (blue). (MOV 473 kb)

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). (MOV 584 kb)

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). (MOV 944 kb)

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Chen, X., Shen, Y., Draper, W. et al. ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.Nat Methods 13, 1013–1020 (2016). https://doi.org/10.1038/nmeth.4031

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• Received: 22 August 2016
• Accepted: 19 September 2016
• Published: 17 October 2016
• Issue date: December 2016
• DOI: https://doi.org/10.1038/nmeth.4031