Simultaneous quantification of protein–DNA interactions and transcriptomes in single cells with scDam&T-seq
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 c...
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| Published in | Nature protocols Vol. 15; no. 6; pp. 1922 - 1953 |
|---|---|
| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
01.06.2020
Nature Publishing Group |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1754-2189 1750-2799 1750-2799 |
| DOI | 10.1038/s41596-020-0314-8 |
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| 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 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.
This protocol combines two genome-wide technologies, DamID and CEL-Seq2, to simultaneously profile DNA interactions with a POI and transcriptomes in single cells. |
|---|---|
| AbstractList | 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 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. 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 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. This protocol combines two genome-wide technologies, DamID and CEL-Seq2, to simultaneously profile DNA interactions with a POI and transcriptomes in single cells. 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 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. This protocol combines two genome-wide technologies, DamID and CEL-Seq2, to simultaneously profile DNA interactions with a POI and transcriptomes in single cells. 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 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.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 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. |
| Audience | Academic |
| Author | de Vries, Sandra S. Lochs, Silke J. A. de Luca, Kim L. Dey, Siddharth S. Markodimitraki, Corina M. Mooijman, Dylan Kind, Jop Rooijers, Koos Rang, Franka J. Chialastri, Alex |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/32350457$$D View this record in MEDLINE/PubMed |
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| Snippet | Protein–DNA interactions are essential for establishing cell type–specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a... Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a... |
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| SubjectTerms | 631/337/100 631/337/2019 631/61/212/177 Adenine Amplification Analytical Chemistry Animals Biological Techniques Biomedical and Life Sciences Cell Line Cell Line, Tumor Cell lines Chromatin Computational Biology/Bioinformatics Deoxyribonucleic acid DNA DNA - genetics DNA - metabolism DNA barcoding DNA Methylation DNA methyltransferase DNA sequencing E coli Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Flow cytometry Fusion protein Gene expression Gene Expression Profiling - methods Genetic research Genetic transcription Genomes Genomics - methods Humans Identification methods Interactomes Life Sciences Methods Mice Microarrays Nucleotide sequencing Organic Chemistry Protein Binding Proteins Proteins - genetics Proteins - metabolism Protocol Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Sequence Analysis, DNA - methods Single-Cell Analysis - methods Site-specific DNA methyltransferase (adenine-specific) Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism Tethering Transcription Transcriptome Transcriptomes |
| Title | Simultaneous quantification of protein–DNA interactions and transcriptomes in single cells with scDam&T-seq |
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