Freedom of expression: cell‐type‐specific gene profiling

Otsuki, Leo; Cheetham, Seth W.; Brand, Andrea H.

doi:10.1002/wdev.149

Cited by 9 publications

(9 citation statements)

References 85 publications

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“…This method requires no cell-type specific purification but involves expressing the protein of interest at high levels, which may in turn influence cell fate or result in aberrant fusion-protein binding. Cell-type specific transcriptional profiling can also be performed in this manner 21 , via the targeted expression of a tagged RNA-binding protein 22 , a ribosomal protein 23 , or an RNA modifying enzyme 24 . These methods often require large amounts of starting material and immunoprecipitation.…”

Section: Comparisons With Other Methodsmentioning

confidence: 99%

“…The digested DNA is column purified to exclude un-cut, genomic DNA from uninduced cells (steps [12][13][14], and adaptors for PCR-amplification are ligated to the DpnI cut fragments (steps [15][16][17][18]. To prevent unmethylated regions of DNA from being aberrantly amplified, the material is digested with DpnII, which only cuts non-methylated GATC sites (steps [19][20][21]). The resulting DNA is then used as template for a PCR reaction to amplify the methylated fragments (steps [22][23][24][25].…”

Section: Overview Of the Proceduresmentioning

confidence: 99%

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Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

et al. 2016

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SummaryThe ability to profile transcription and chromatin binding in a cell type-specific manner is a powerful approach for understanding cell fate specification and cellular function in multicellular organisms.We recently developed Targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA-and chromatin-binding proteins in vivo without cell isolation. As a Protocol Extension, this article describes substantial modifications to an existing Protocol and offers additional applications.TaDa builds upon DamID, a technique for detecting genome-wide DNA binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution.TaDa ensures that Dam-fusion proteins are expressed at very low levels, avoiding toxicity and potential artefacts from over-expression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to Nextgeneration Sequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared to other methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II, TaDa can also identify transcribed genes in a cell type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure -from collecting tissue samples to generating sequencing libraries -can be accomplished within 5 days.

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Section: Comparisons With Other Methodsmentioning

confidence: 99%

Section: Overview Of the Proceduresmentioning

confidence: 99%

Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

et al. 2016

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“…As a result, mapping transcription-factor occupancy in vivo in rare cell types, such as stem cells, is technically challenging. DNA adenine methylation identification (DamID) has recently emerged as an alternative approach for genome-wide profiling (Marshall and Brand, 2015;Marshall et al, 2016;Otsuki et al, 2014;Southall et al, 2013;van Steensel and Henikoff, 2000). In DamID, a DNA-or chromatin-binding protein is fused to an E. coli Dam methylase.…”

Section: Introductionmentioning

confidence: 99%

Targeted DamID reveals differential binding of mammalian pluripotency factors

et al. 2018

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The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.

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“…Each human organ consists of many different types of cells working together to carry out complex physiological functions, but understanding cell-type-specific gene expression (Otsuki et al, 2014) is challenging because most published human transcriptome datasets are composite gene-expression profiles generated from mixed cell populations. In this issue of Cell Systems , Butler et al (2016) report a relatively simple but potentially effective way to identify endothelial-cell-enriched genes based on the straightforward correlation of three endothelial-cell-specific reference genes with transcriptome datasets generated from unfractionated human tissues (Butler et al, 2016).…”

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confidence: 99%