Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq
Markodimitraki, Corina M; Rang, Franka J; Rooijers, Koos; de Vries, Sandra S; Chialastri, Alex; de Luca, Kim L; Lochs, Silke J A; Mooijman, Dylan; Dey, Siddharth S; Kind, Jop
(2020) Nature protocols, volume 15, issue 6, pp. 1922 - 1953
(Article)
Abstract
Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the
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tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.
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Keywords: Animals, Cell Line, Cell Line, Tumor, DNA Methylation, DNA/genetics, Escherichia coli Proteins/genetics, Escherichia coli/genetics, Gene Expression Profiling/methods, Genomics/methods, Humans, Mice, Protein Binding, Proteins/genetics, Recombinant Fusion Proteins/genetics, Sequence Analysis, DNA/methods, Single-Cell Analysis/methods, Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics, Transcriptome, General Biochemistry,Genetics and Molecular Biology, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Journal Article, Research Support, N.I.H., Extramural
ISSN: 1750-2799
Publisher: Nature Publishing Group
Note: Funding Information: We thank the members of the Kind laboratory for their comments on the manuscript. S.S.D. acknowledges support from the Center for Scientific Computing at UCSB: an NSF MRSEC (DMR-1720256) and NSF CNS-1725797. S.S.D. was also supported by the NIH grant R01HG011013. This work was funded by a European Research Council Starting grant (ERC-StG 678423-EpiID), a Nederlandse Orga-nisatie voor Wetenschappelijk Onderzoek37 Open (824.15.019) and ALW/VENI grant (016.181.013). The Oncode Institute is supported by KWF Dutch Cancer Society. Publisher Copyright: © 2020, The Author(s), under exclusive licence to Springer Nature Limited. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
(Peer reviewed)