Improved methods for deamination-based m6A detection
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA804865 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17956459_1.fastq.gz | Fastqsanger.gz | |||
| SRR17956459_2.fastq.gz | Fastqsanger.gz | |||
| SRR17956460_1.fastq.gz | Fastqsanger.gz | |||
| SRR17956460_2.fastq.gz | Fastqsanger.gz | |||
| SRR17956461_1.fastq.gz | Fastqsanger.gz |
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Project description:We present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition in APO1-YTH (D422N). In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA.
2022-05-11 | GSE196416 | GEO
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.
2022-10-24 | GSE201062 | GEO
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.
2022-10-24 | GSE201063 | GEO
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.
2022-10-24 | GSE211303 | GEO
Project description:m6A is a widespread RNA modification which plays important roles in the regulation of gene expression. Methods for the global detection of m6A rely on immunoprecipitation of methylated transcripts using m6A antibodies. However, these methods are costly and require large amounts of input RNA, making them prohibitive for many experiments. Here, we describe DART-seq, an antibody-free method for m6A detection which enables transcriptome-wide mapping of m6A residues using low amounts of input material. DART-seq can be used to obtain global m6A maps using as little as 10 nanograms of total RNA and at single-molecule resolution. This method offers several improvements over current techniques and will facilitate detection of m6A in limiting cell and tissue types.
2019-09-23 | GSE125780 | GEO
Project description:N6-Methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of the high throughput detection of methylated sites in mRNA using MeRIPSeq or miCLIP revolutionized the m6A research field. Both methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. Currently such approaches are limited. Here we present RedBaron, an improved biochemical method for the site-specific detection and quantification of m6A in RNA. We demonstrate that the RedBaron method is able to accurately quantify m6A levels at specific transcripts in vivo. We used this assay for the site-specific detection and quantification of m6A within the chicken β-actin (ACTB) zipcode sequence in chicken embryos and in fibroblast cells. We demonstrate that methylation of this site in the β-actin zipcode enhances ZBP1 binding in vitro, whilst methylation of a nearby adenosine abolishes
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Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
2023-07-12 | E-MTAB-9542 | biostudies-arrayexpress
Project description:Transcriptome-wide deamination analysis
| PRJNA951692 | ENA
Project description:N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate and function. Current m6A mapping approaches rely on immunoprecipitation of m6A-containing RNA fragments to identify regions of transcripts that contain m6A. This approach localizes m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here we show that anti-m6A antibodies can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Similarly, we find these antibodies induce mutational signatures at N6, 2’-O-dimethyladenosine (m6Am), a nucleotide found at the first encoded position of certain mRNAs. Using these mutational signatures, we map m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identify snoRNAs as a novel class of m6A-containing ncRNAs. UV-crosslinking and immunoprecipitation with m6A-specific antibodies was used to map m6A and m6Am in cellular RNA with single nucleotide resolution.
2015-06-29 | E-GEOD-63753 | biostudies-arrayexpress
Project description:5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that can regulate gene expression. While some methods were developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution are still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method for the genome-wide mapping of 5hmC at single-base resolution. This method capitalizes on a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine (C), 5-methylcytosine (5mC), and 5hmC. In SSD-seq, an engineered A3A protein (eA3A-v10) can adequately deaminate C and 5mC, but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A-v10 to form uracil (U) and thymine (T), both of which are read as T during sequencing. However, 5hmC is resistant to the deamination by eA3A-v10 and is still read as C during sequencing. Therefore, the remaining C in the sequence reads manifests the original 5hmC. Applying SSD-seq to generate a base-resolution map of 5hmC in human lung tissue, we found that 5hmC was almost entirely confined to CpG dinucleotides. The base-resolution map of 5hmC from human lung tissue generated by SSD-seq correlated strongly with that generated by prior ACE-seq. Taken together, the SSD-seq method is single-step, bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the direct and quantitative detection of 5hmC in genomes at single-base resolution.
2023-12-04 | GSE236353 | GEO