Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells

Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.

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“…The amplified RNA from IVT is then used to prepare Illumina sequencing libraries, as described previously ( Fig. 1 and Methods) [20][21][22].…”

Section: Embr-seq Uses Blocking Primers To Deplete Rrnamentioning

confidence: 99%

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

et al. 2020

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“…It still needs to be addressed whether the differential transcriptional sensitivities observed in these studies may be related to differences in flanking chromatin signatures or to the properties of adjacent LADs (cLADs or fLADs). A recent study, employing a new method to simultaneously measure LADs and mRNA from the same single cell, may provide a platform to systematically decipher the molecular interactions causing the differences in transcriptional dependencies for individual LADs in various cell types [49].…”

Section: Lads and The Regulation Of Gene Expressionmentioning

confidence: 99%

“…This apparent discrepancy might be explained by differences in the proportion of LADs that associate with the NL in individual cells. Indeed, while cells in a more undifferentiated state show more heterogeneity between LAD content of individual cells [10,49], terminally differentiated single cells may have a higher proportion of LADs consistently contacting the NL, perhaps due to the increase in H3K9me3 levels. Interestingly, inhibition of H3K9 methylation results in a differentiation delay [62] and, removal of H3K9me3 alleviates binding restrictions of pluripotency factors to DNA and facilitates reprogramming [63].…”

Section: Lads and The Regulation Of Gene Expressionmentioning

confidence: 99%

Spatial chromatin organization and gene regulation at the nuclear lamina

Guerreiro

Kind

2019

Current Opinion in Genetics & Development

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The nuclear lamina (NL) consists of a thin meshwork of lamins and associated proteins that lines the inner nuclear membrane (INM). In metazoan nuclei, a large proportion of the genome contacts the NL in broad lamina-associated domains (LADs). Contacts of the NL with the genome are believed to aid the spatial organization of chromosomes and contribute to the regulation of transcription. Here, we will focus on recent insights in the structural organization of the genome at the NL and the role of this organization in the regulation of gene expression.

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“…ChIP-seq is the most popular technique for studying TF binding [25], and while single cell ChIP-seq has been previously described [26], this technique has only been employed to map highly abundant proteins such as methylated histones. DamID can recover TF binding sites by identifying nearby exogenously methylated adenines [27,28], but in single cells it has only been used to study laminin-associated domains [29][30][31]. Importantly, both methods yield sparse data, either in small numbers of cells [31] or without simultaneously capturing mRNA [26].…”

Section: Introductionmentioning

confidence: 99%

“…DamID can recover TF binding sites by identifying nearby exogenously methylated adenines [27,28], but in single cells it has only been used to study laminin-associated domains [29][30][31]. Importantly, both methods yield sparse data, either in small numbers of cells [31] or without simultaneously capturing mRNA [26]. Thus, each can only be used in heterogeneous samples if the cell type is known a priori and if sufficient numbers of cells are obtained by selection or sorting to overcome sparsity.…”

Section: Introductionmentioning

confidence: 99%

Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

Moudgil

Wilkinson

Chen

et al. 2019

Preprint

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In situ assays of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

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Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells

Abstract: 20

Cited by 22 publications

References 35 publications

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

Spatial chromatin organization and gene regulation at the nuclear lamina

Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

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