A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility

Xu, Wei; Wen, Yi; Liang, Yingying; Xu, Qiushi; Wang, Xuefei; Jin, Wenfei; Chen, Xi

doi:10.1038/s41596-021-00583-5

“…62248) was added at a final concentration of 1 μg/μl to stain the nuclei. DAPI positive single nuclei were sorted into each well in a 384-well plate containing 3 μl lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM sodium chloride, 0.2% SDS, 10 μM N7xx primer, 10 μM Truseq_S5_short primer, 10 μM Nextra_S5_short primer) by FACS as previously described 34 . The plates were incubated at 65 °C, 15 min in a thermocycler for cell lysis.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were demultiplexed using deML 48 . For the plate-based workflow ATAC-seq data, Read 1 and Read 2 FastQ files were processed using the Snakemake 49 pipeline as described previously 34 , except that BWA 50 was used for the alignment. For the plate-based workflow RNA-seq data, the combination of i7 and i5 defines a single cell and the first 10 bp of Read 1 were UMIs.…”

Section: Methodsmentioning

confidence: 99%

ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells

Xu

¹

,

Yang

²

,

Zhang

³

et al. 2022

Preprint

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Joint profiling of chromatin accessibility and gene expression from the same single cell/nucleus provides critical information about cell types in a tissue and cell states during a dynamic process. These emerging multi-omics techniques help the investigation of cell-type resolved gene regulatory mechanisms. However, many methods are currently limited by low sensitivity, low throughput or complex workflow. Here, we developed in situ SHERRY after ATAC-seq (ISSAAC-seq), a highly sensitive and flexible single cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single nucleus. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from over 10,000 nuclei from the mouse cerebral cortex, we uncovered major and rare cell types and cell-type specific regulatory elements and identified heterogeneity at the chromatin level within established cell types defined by gene expression. Finally, we revealed distinct dynamics and relationships of gene expression and chromatin accessibility during an oligodendrocyte maturation trajectory.

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“…DAPI (Thermo Fisher) was added at a final concentration of 1 μg/μl to stain the nuclei. DAPI positive single nuclei were sorted into each well in a 384-well plate containing 3 µl lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM sodium chloride, 0.2% SDS, 10 μM N7xx primer, 10 μM Truseq_S5_short primer, 10 μM Nextra_S5_short primer) by FACS as previously described 34 . The plates were incubated at 65 °C, 15 min in a thermocycler for cell lysis.…”

Section: Single Nuclei Sorting and Library Pre-amplification In Plate...mentioning

confidence: 99%

“…Here we developed ISSAAC-seq, a highly sensitive and scalable method to interrogate chromatin accessibility and gene expression from the same single nucleus with a flexible workflow. The method is based on the combination of the recently developed Sequencing HEteRo RNA-DNA-hYbrid (SHERRY) 32,33 and previously established scATAC-seq approaches 16,19,20,34 (Fig. 1a, Supplementary Fig.…”

mentioning

confidence: 99%

ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells

Xu

¹

,

Yang

²

,

Zhang

³

et al. 2022

Self Cite

View full text Add to dashboard Cite

Joint profiling of chromatin accessibility and gene expression from the same single cell/nucleus provides critical information about cell types in a tissue and cell states during a dynamic process. These emerging multi-omics techniques help the investigation of cell-type resolved gene regulatory mechanisms 1-7 . However, many methods are currently limited by low sensitivity, low throughput or complex workflow. Here, we developed in situ SHERRY after ATAC-seq (ISSAAC-seq), a highly sensitive and flexible single cell multi-omics method to interrogate chromatin accessibility and gene expression from the same single nucleus. We demonstrated that ISSAAC-seq is sensitive and provides high quality data with orders of magnitude more features than existing methods. Using the joint profiles from over 10,000 nuclei from the mouse cerebral cortex, we uncovered major and rare cell types and cell-type specific regulatory elements and identified heterogeneity at the chromatin level within established cell types defined by gene expression. Finally, we revealed distinct dynamics and relationships of gene expression and chromatin accessibility during an oligodendrocyte maturation trajectory. Main TextRecent technological development enables detailed characterization of various modalities at the single cell level, such as gene expression [8][9][10][11][12][13] , chromatin accessibility [14][15][16][17][18][19][20] and protein abundance 21,22 . Data from different modalities provide distinct and complementary information about cell types or states. Currently, most methods only assay one modality of the cell at a time. Though highly informative, only one molecular layer of the cell is profiled, .

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“…33 Currently, both plate-based (interrogation of hundreds of cells) and droplet-based (interrogation of tens of thousands of cells) methods for scATAC-seq are available. 34,35 Additionally, DNA-protein interactions in single cells can be investigated via chromatin immunoprecipitation sequencing (ChIP-seq). 36 This method identifies binding sites of DNA-associated proteins.…”

Section: Chromatin Statementioning

confidence: 99%

Single‐cell omics: Overview, analysis, and application in biomedical science

Stein

¹

,

Weiskirchen

²

,

Damm

³

et al. 2021

J of Cellular Biochemistry

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Single-cell sequencing methods provide the highest resolution insight into cellular heterogeneity. Owing to their rapid growth and decreasing cost, they are now widely accessible to scientists worldwide. Single-cell technologies enable analysis of a large number of cells, making them powerful tools to characterise rare cell types and refine our understanding of diverse cell states. Moreover, single-cell application in biomedical sciences helps to unravel mechanisms related to disease pathogenesis and outcome. In this Viewpoint, we briefly describe existing single-cell methods (genomics, transcriptomics, epigenomics, proteomics, and mulitomics), comment on available analysis tools, and give examples of method applications in the biomedical field.

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A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility

Cited by 45 publications

References 67 publications

ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells

ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells

ISSAAC-seq enables sensitive and flexible multimodal profiling of chromatin accessibility and gene expression in single cells

Single‐cell omics: Overview, analysis, and application in biomedical science

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