Single base mapping of m6A by an antibody-independent method
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA515628 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:N6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, related to pivotal RNA metabolism processes. The most popular high-throughput m6A identification method relies on the commercial m6A antibody but suffers from poor reproducibility and limited resolution. Exact location of m6A site is of great vital for understanding the dynamics, functions and machinery of RNA methylation. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic single base m6A map generated by m6A-REF-seq displayed a typical distribution pattern with enrichment adjacent to stop codon. Ligase-based and qPCR validation methods were used to confirm the individual m6A sites and quantify the methylation level, reinforcing the high accuracy of m6A-REF-seq. We applied m6A-REF-seq on five tissues from three mammals, showing that m6A sites were conserved and tend to gather together among species. (m6A-REF-seq had been named as Aim-seq.)
2019-07-03 | GSE125240 | 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
2023-05-24 | GSE185078 | 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:DART-seq: an antibody-free method for global m6A detection
| PRJNA517490 | 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:Here we report a metabolic labeling method to map mRNA N6-methyladenosine (m6A) modification transcriptome-wide at base resolution, termed m6A-label-seq. The cells were fed with Se-allyl-L-selenohomocysteine, an analog of methoine, which serves as the precursor of methylation enzyme cofactor, so that cellular RNAs were continuously deposited with N6-allyladenosine (a6A) at supposed m6A sites. We enriched a6A-containing mRNAs and sequenced their a6A sites which are identical to m6A sites, based on iodination-induced misincorporation during reverse transcription.
2020-04-21 | GSE131316 | GEO
Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing. Identification of m6A modified sequences in HepG2 cells.
2012-04-25 | E-GEOD-37003 | biostudies-arrayexpress
Project description:We developed a quantitative method called GLORI to investigate m6A methylation in the mammalian transcriptome at single-base resolution.
2023-06-18 | GSE233875 | GEO
Project description:To investigate the m6A methylation in the mammalian transcriptome, we developed a quantitative method, names GLORI, that could detect m6A stoichiometry at single-base resolution. We then performed GLORI on cells with different treatment, such as stress, knockdown and inhibitor. We next analysis m6A methyloms of different celllines and cells under different treatment to investigate the fuctional role of m6A.
2022-08-06 | GSE210563 | GEO
Project description:We developed a novel approach, m6A-seq, for high-resolution mapping of the transcriptome-wide m6A landscape, based on antibody-mediated capture followed by massively parallel sequencing Identification of m6A modified sequences in mouse liver and human brain
2012-04-25 | E-GEOD-37004 | biostudies-arrayexpress