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Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

ABSTRACT: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

PROVIDER: PRJNA300405 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR2846090.fastq.gz	Fastqsanger.gz
	SRR2846091.fastq.gz	Fastqsanger.gz
	SRR2846086_1.fastq.gz	Fastqsanger.gz
	SRR2846086_2.fastq.gz	Fastqsanger.gz
	SRR2846087_1.fastq.gz	Fastqsanger.gz

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Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

Project description:To interrogate single-base resolution 6mA sites in the genome-wide, we develop DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an optimized sequencing method taking advantage of restriction enzyme DpnI, which exclusively cleaves methylated adenine sites. We find DpnI also recognizes other sequence motifs besides the canonical GATC restriction sites, largely expanding the application range of this method. DA-6mA-seq requires less starting material and lower sequencing depth than previous methods, but achieves higher sensitivity, providing a good strategy to identify 6mA in large genome with a low abundance of 6mA. We rebuild the 6mA maps of Chlamydomonas by DA-6mA-seq and then apply this method to another two eukaryotic organisms, Plasmodium and Penicillium. Further analysis reveals most 6mA sites are symmetric at various sequence contexts, suggesting 6mA may function as a new heritable epigenetic mark in eukaryotes. A new sequencing method is developed to detect 6mA in eukaryotes

2016-04-12 | E-GEOD-74430 | biostudies-arrayexpress

Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

2016-04-12 | GSE74430 | GEO

Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.

Project description:Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

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Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine

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Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [site_specific_quantification]

Project description:We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome.

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Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [probes_assay]

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Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [eTAM-seq]

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Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine

Project description:We applied chemical-assisted, m6A-SEAL method to profile m6A distributions in the transcriptomes of human and plant cells and compared its results against MeRIPm6A-seq.

2020-04-22 | GSE129979 | GEO

Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination

Project description:Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination

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