An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

Corces, M. Ryan; Trevino, Alexandro E.; Hamilton, Emily G.; Greenside, Peyton G.; Sinnott‐Armstrong, Nicholas A.; Vesuna, Sam; Satpathy, Ansuman T.; Rubin, Adam J.; Montine, Kathleen S.; Wu, Beijing; Kathiria, Arwa S.; Cho, Seung Woo; Mumbach, Maxwell R.; Carter, Ava C.; Kasowski, Maya; Orloff, Lisa A.; Risca, Viviana I.; Kundaje, Anshul; Khavari, Paul A.; Montine, Thomas J.; Greenleaf, William J.; Chang, Howard Y.

doi:10.1038/nmeth.4396

Cited by 1,963 publications

(1,581 citation statements)

References 26 publications

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“…Of note, cell-line-specific TFs were also detected (such as SOX11 in A375), and each melanoma culture contains cells with increased activity of cell cycle regulons such as FOXM1 (Figure 3b). Next, to validate our findings, we profiled the chromatin landscape of these nine MM lines, using Omni-ATAC-seq (Corces et al 2017). As expected, the melanocyte-and mesenchymallike cultures display preferential accessibility of the previously-identified state-specific H3K27Ac regions (Verfaillie et al 2015) (Supplementary Figure S4b).…”

Section: Single-cell Network Inference Reveals Candidate Regulators Osupporting

confidence: 60%

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

Wouters

Atak

Minnoye

et al. 2019

Preprint

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Melanoma is notorious for its cellular heterogeneity, which is at least partly due to its ability to transition between alternate cell states. Similarly to EMT, melanoma cells with a melanocytic phenotype can switch to a mesenchymal-like phenotype. However, scattered emerging evidence indicates that additional, intermediate state(s) may exist. In order to search for such new melanoma states and decipher their underlying gene regulatory network (GRN), we extensively studied ten patient-derived melanoma cultures by single-cell RNA-seq of >39,000 cells. Although each culture exhibited a unique transcriptome, we identified shared gene regulatory networks that underlie the extreme melanocytic and mesenchymal cell states, as well as one (stable) intermediate state. The intermediate state was corroborated by a distinct open chromatin landscape and governed by the transcription factors EGR3, NFATC2, and RXRG. Single-cell migration assays established that this "transition" state exhibits an intermediate migratory phenotype. Through a dense time-series sampling of single cells and dynamic GRN inference, we unraveled the sequential and recurrent arrangement of transcriptional programs at play during phenotype switching that ultimately lead to the mesenchymal cell state. We provide the scRNA-Seq data with 39,263 melanoma cells on our SCope platform and the ATAC-seq data on a UCSC hub to jointly serve as a resource for the melanoma field. Together, this exhaustive analysis of melanoma cell state diversity indicates that additional states exists between the two extreme melanocytic and mesenchymal-like states. The GRN we identified may serve as a new putative target to prevent the switch to mesenchymal cell state and thereby, acquisition of metastatic and drug resistant potential.

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Section: Single-cell Network Inference Reveals Candidate Regulators Osupporting

confidence: 60%

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

Wouters

Atak

Minnoye

et al. 2019

Preprint

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“…Tissue sections were snap frozen and stored at -80C. Nuclei were isolated from frozen tissues using the protocol published in Corces MR et al, 2017 65 . Briefly, frozen tissue samples were thawed in 2mL chilled Homogenization Buffer (10mM Tris pH7.8, 5mM CaCl2, 3mM Mg(Ac)2, 320 mM Sucrose, 0.1mM EDTA, 0.1% NP40, 167uM βmercaptoethanol, 16.7uM PMSF) and lysed in a pre-chilled dounce.…”

Section: Nuclei Isolation From Frozen Primary Tissuementioning

confidence: 99%

Single cell epigenomic atlas of the developing human brain and organoids

Rs¹,

Wilfert

et al. 2019

Preprint

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31Dynamic changes in chromatin accessibility coincide with important aspects of neuronal differentiation, such as 32 fate specification and arealization and confer cell type-specific associations to neurodevelopmental disorders. 33However, studies of the epigenomic landscape of the developing human brain have yet to be performed at single-34 cell resolution. Here, we profiled chromatin accessibility of >75,000 cells from eight distinct areas of developing 35human forebrain using single cell ATAC-seq (scATACseq). We identified thousands of loci that undergo 36 extensive cell type-specific changes in accessibility during corticogenesis. Chromatin state profiling also reveals 37 novel distinctions between neural progenitor cells from different cortical areas not seen in transcriptomic profiles 38 and suggests a role for retinoic acid signaling in cortical arealization. Comparison of the cell type-specific 39 chromatin landscape of cerebral organoids to primary developing cortex found that organoids establish broad 40 cell type-specific enhancer accessibility patterns similar to the developing cortex, but lack many putative 41 regulatory elements identified in homologous primary cell types. Together, our results reveal the important 42 contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and 43 provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical 44 development. 45 46Main text 47The diverse cell types of the human cerebral cortex (Fig. 1a) have been mostly classified based on a handful of 48 morphological, anatomical, and physiological features. Recent innovations in single cell genomics, such as single 49 cell mRNA sequencing (scRNA-seq), have enabled massively parallel profiling of thousands of molecular 50 features in every cell, uncovering the remarkable molecular diversity of cell types previously considered 51 homologous, such as excitatory neurons located in different areas of the cerebral cortex 1-6 . However, the 52 developmental mechanisms underlying the emergence of distinct cellular identities are largely unknown, as most 53 cortical neurons are generated at stages that are inaccessible to experimentation 5 . 54 55Over 60 years ago, Conrad Waddington introduced the concept of an epigenomic landscape to account for the 56 emergence of distinct cell fates 7 . In particular, chromatin state defines the functional architecture of the genome 57

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“…To evaluate SNARE-seq's ability to capture accessible chromatin, we first performed a proof-ofconcept experiment on lymphoblastoid cell line GM12878, which have extensively characterized chromatin landscapes. Ensemble profiles of SNARE-seq accessibility data showed a signal-tonoise ratio similar to ATAC-seq 6 and Omni-ATAC 7 (Fig. 1b).…”

mentioning

confidence: 65%

Linking transcriptome and chromatin accessibility in nanoliter droplets for single-cell sequencing

Chen

Lake

Zhang

2019

Preprint

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Linked profiling of transcriptome and chromatin accessibility from single cells can provide unprecedented insights into cellular status. Here we developed a droplet-based Single-Nucleus chromatin Accessibility and mRNA Expression sequencing (SNARE-seq) assay, that we used to profile neonatal and adult mouse cerebral cortices. To demonstrate the strength of single-cell dual-omics profiling, we reconstructed transcriptome and epigenetic landscapes of cell types, uncovered lineage-specific accessible sites, and connected dynamics of promoter accessibility with transcription during neurogenesis.RNA sequencing of single cells or nuclei can reveal their transcription state, while chromatin accessibility sequencing would uncover the associated upstream transcriptional regulatory landscape. Current strategies 1,2 , which involve profiling these modalities separately followed by computational integration, may not fully recapitulate the true biological state. Joint profiling of two layers of -omics information within the same cells would enable a direct matching of transcriptional regulation to its output, allowing for more accurate reconstruction of the molecular processes underlying a cell's physiology. To enable highly parallel profiling of chromatin accessibility and mRNA from individual nuclei, we developed SNARE-seq, implemented on a micro-droplet platform 3 . In this method, the accessible chromatin in permeabilized nuclei are captured by the Tn5 transposase, prior to droplet generation. We reason that, without heating or detergent treatment, binding of transposases to its DNA substrate after transposition could maintain the contiguity of the original DNA strands 4 , allowing for the copackaging of accessible genomic sites and mRNA from individual nuclei in the same droplets.

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An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

Cited by 1,963 publications

References 26 publications

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

Single cell epigenomic atlas of the developing human brain and organoids

Linking transcriptome and chromatin accessibility in nanoliter droplets for single-cell sequencing

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