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Robust bisulfite free SMRT Sequencing of mdC based on a novel hpTet3-enzyme

ABSTRACT: Robust bisulfite free SMRT Sequencing of mdC based on a novel hpTet3-enzyme

PROVIDER: PRJNA1079692 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28068104_subreads.fastq.gz	Fastqsanger.gz
	SRR28068105_subreads.fastq.gz	Fastqsanger.gz
	SRR28068106_subreads.fastq.gz	Fastqsanger.gz
	SRR31146165_1.fastq.gz	Fastqsanger.gz
	SRR31146165_2.fastq.gz	Fastqsanger.gz

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Robust bisulfite free SMRT Sequencing of mdC based on a novel hpTet3-enzyme

Project description:Genomic DNA contains next to the four canonical nucleosides dA, dC, dG and T the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies has an instrumental effect on the transcriptional activity of the corresponding gene. The methylation patterns of genes are therefore responsible for either silencing or activation of genes. Sequencing of mdC positions in the genome is consequently of paramount importance for early cancer diagnostics in order to determine incorrect expression of genes. Today, the bisulfite method is the gold standard for mdC-sequencing, which has however the caveat that the majority of the input DNA is degraded during the bisulfite treatment. In addition, bisulfite sequencing is rather error prone. Here we report a benign, bisulfite free mdC sequencing method based on 3rd generation single molecule SMRT sequencing. The fundament of the technology is a new Tet3 enzyme that oxidizes mdCs with high efficiency to 5-carboxycytidine (cadC). caC in turn provides and excellent read out by SMRT sequencing using specially trained AI-based algorithms.

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DNA methylation estimation using methylation-sensitive restriction enzyme bisulfite sequencing (MREBS)

Project description:We introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. Here, we compare MREBS to WGBS and RRBS.

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Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

Project description:5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow the m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

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A bisulfite‐free approach for base‐resolution analysis of genomic 5-carboxylcytosine

Project description:Due to an extreme rarity of 5-carboxylcytosine (5caC) in mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental/computational resources increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high resolution mapping of 5caCGs. The method employs an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC generating unmodified CGs which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supported the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflected their distinct demethylation dynamics and demonstrated an association of 5caC with gene expression. Generally, we demonstrated that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC.

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Project description:The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor that regulates the transcriptional activity of many transcription factors critical for metabolic processes. While the importance of SMRT’s role in the adipocyte has been well-established, prior mouse models have yielded contradictory phenotypes, limiting our understanding of its in vivo function in the context of homeostatic maintenance. Multiple models suggest that SMRT deficiency leads to increased adiposity, though the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define SMRT’s metabolic contributions. In doing so, we found that SMRT deletion in the adipocyte does not, in fact, lead to obesity, despite increased food consumption in knockouts – even when mice are challenged with a high-fat diet. This suggests that prior adiposity phenotypes described in generalized models were due to effects beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious effect was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Taken together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to effectively maintain physiological homeostasis.

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