Epitranscriptomic sequencing

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depend on the incorporation of photoactivatable ribonucleosides - the antibody directly crosslinks with a base close (very predictable location) to the m6A site. These UV-based strategies uses antibodies that induces consistent and predictable mutational and truncation patterns in the cDNA strand during reverse-transcription that could be leveraged to more precisely locate the m6A site. Though both m6A-CLIP and miCLIP reply on UV induced mutations, m6A-CLIP is distinct by taking advantage that m6A alone can induce cDNA truncation during reverse transcription and generate single-nucleotide mapping for over ten folds more precise m6A sites (MITS, m6A-induced truncation sites), permitting comprehensive and unbiased precise m6A mapping. In contrast, UV-mapped m6A sites by miCLIP is only a small subset of total precise m6A sites. The precise location of tens of thousands of m6A sites in human and mouse mRNAs by m6A-CLIP reveals that m6A is enriched at last exon but not around stop codon.

967:: when a modified base is present, the biophysical dynamics of its movement changes, creating a unique kinetic signature before, during, and after the base incorporation. SMRT sequencing can be used to detect modified bases in RNA, including m6A sites. In this case, a reverse transcriptase is used as enzyme with ZMWs to observe the cDNA synthesis in real time. The incorporation of synthetically designed m6A sites leaves a kinetic signature and increases the interpulse duration (IPD). There are some issues concerning the reading of homonucleotide stretches and the base resolution of m6A therein, due to the stuttering of reverse transcriptase. Secondly, the throughput is too low for transcriptome-wide approaches. One of the most commonly used platform is the SMRT sequencing technology by 890:→ implicates the selective labelling of a specific position within the sequence: these techniques rely on the Stanley-Vassilenko approach principles, that has been adjusted to achieve a better validation quality. First, RNA is cleaved into free 5’-OH fragments either by RNase H or DNAzymes, by sequence specific hydrolysis. The polynucleotide kinase (PKN) then performs the 5’ radioactive post-labelling phosphorylation using ATP. At this point, the labelled fragments undergo a size fragmentation, that can be performed either by 681:

inosine-specific cleavage, in that if there are two A-to-I modifications in relatively close proximity, the downstream mod is less likely to be detected since the cDNA synthesis will be truncated at a prior nucleotide. Both ICE and ICE-seq suffer from a lack of sensitivity to infrequently edited locations: it becomes difficult to distinguish a modification with a frequency of <10% from a false positive. An increase in read depth and quality can increase sensitivity, but also then suffer from further amplification bias.

500:(Roche) for analyzing deep sequencing data to quantify sequence-specific cytosine content. However, recent papers have suggested that the method have several flaws: (1) Incomplete conversion of regular cytosines in double-stranded regions of RNA; (2) areas containing other modifications that resulted in bisulfite-treatment resistance; and (3) sites containing potential false-positives due to (1) and (2) In addition, it is possible the sequencing depth is still not high enough to correctly detect all methylated sites. 163:

elution and collection of antibody-tagged RNA molecules. The immunoprecipitation procedure in MeRIP-Seq is able to produce >130fold enrichment of m6A sequences. Random primed cDNA library generation was performed, followed by adaptor ligation and Illumina sequencing. Since the RNA strands are randomly chopped up, the m6A site should, in principle, lie somewhere in the center of the regions to which sequence reads align. At extremes, the region would be roughly 200nt wide (100nt up- and downstream of the m6A site).

220:, followed by finding the consensus methylation sequence. The presence of the T to C mutation helps increase the signal to noise ratio of methylation site detection as well as providing greater resolution to the methylation sequence. One shortcoming of this method is that m6A sites that did not incorporate 4SU can't be detected. Another caveat is that position of 4SU incorporation can vary relative to any single m6A residue, so it still remains challenging to precisely locate m6A site using the T to C mutation. 984:

leads to a disruption in current stream, which is different for the different bases, included modified ones, and therefore can be used to identify possible modifications. By producing single-molecule reads, without previous RNA amplification and conversion to cDNA, these techniques can lead to the production of quantitative transcriptome-wide maps. In particular, the Nanopore technology proved to be effective in detecting the presence of two nucleotide analogs in RNA:

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modification profiling. However, there are further functions of ADAR enzymes within the cell — for example, they have further roles in RNA processing, and in miRNA biogenesis — which would also be likely to change the landscape of cellular mRNA. Recently a map of A-to-I editing in mice was generated using editing-deficient ADAR1 and ADAR2 double-knockout mice as a negative control. Thereby, A-to-I editing was detected with high confidence.

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a percentage. This method potentially has single-nucleotide resolution. In fact, the abundance of RNA-seq data that is now publicly available can be leveraged to investigate G (in cDNA) versus A (in genome). One particular pipeline, called RNA and DNA differences (RDD), claims to excludes false positives, but only 56.8% of its A-to-I sites were found to be valid by ICE-seq (see below).

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discovered telomerase RNA component (TERC) RNA, scaRNAs and snoRNAs as new classes of Nm-containing ncRNAs as well as identified many 2'-O-methylation sites in various ncRNAs and mRNAs. Furthermore, Nm-REP-seq revealed 2'-O-Methylation located at the 3’-end of snoRNAs, snRNAs, tRNAs and fragments derived from them, as well as piRNAs and miRNAs.

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preparation protocol capturing both ends of the fragmented RNA, ensuring that the methylated position is within the sequenced fragment. By additionally referencing the m6A consensus motif and eliminating false positive m6A peaks using negative control samples, the m6A profiling in yeast was able to be done at single-base resolution.

880:→ involves the use of P: cells are grown in P containing medium, thus allowing the incorporation of NTPs during transcription by T7 RNA polymerase. The modified RNA is then extracted, and each RNA species is isolated and subsequently digested by T2 RNase. Next, RNA is hydrolyzed into 5' nucleoside monophosphates, which are analyzed 167:

reads tending to pileup at the 5’ terminus of the transcript. Schwartz et al. (2015) leveraged this knowledge to detect mTSS sites by picking out sites with a high ratio of the size of pileups in the IP samples compared to input sample. As confirmation, >80% of the highly enriched pileup sites contained adenosine.

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as well as PIWI-interacting RNAs (piRNAs) in animals.This modification can perturb the function of ribosomes and disrupt tRNA decoding, regulate alternative splicing fidelity, protect ncRNAs from 3’-5’ exonucleolytic degradation and provide a molecular signature for discrimination of self from non-self mRNA.

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SCARLET: this recent approach exploits not just one, but two sequence selection steps, the last of which is obtained during the splinted ligation of the radioactive-labelled fragments with a long DNA oligonucleotide, at its 3’-end. After degradation, the labelled residue is purified together with the

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can therefore be compared to the corresponding genomic sequences; in sites where A residues are repeatedly interpreted as G, a methylation event can be assumed. At high enough accuracy, it is feasible that the quantity of mRNA molecules in the population that have been methylated can be calculated as

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The 2'-O-methylation of the ribose moiety is one of the most common RNA modifications and is present in diverse highly abundant non-coding RNAs (ncRNAs) and at the 5' cap of mRNAs. Moreover, many studies have revealed that Nm at 3’-end is presented in some ncRNAs, such as microRNAs (miRNAs) in plants

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m6A-LAIC-seq (m6A-level and isoform-characterization sequencing) is a high-throughput approach to quantify methylation status on a whole-transcriptome scale. Full-length RNA samples are used in this method. RNAs are first subjected to immunoprecipitation with an anti-m6A antibody. Excess antibody is

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technologies. This technique exploits nanometer-sized protein channels embedded into a membrane or solid materials, and coupled to sensors, able to detect the amplitude and duration of the variations of the ionic current passing through the pore. As the RNA passes through the nanopore, the blockage

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step and therefore required primers and knowledge of the location or regions to be investigated, alongside a maximum cDNA length of 300–500bp. The ICE-seq method is complicated, along with being labour-, reagent- and time-intensive. One protocol from 2015 took 22 days. This shares a limitation with

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is reacted with inosine to form N1-cyanoethylinosine (ce1I). This serves to stall reverse transcriptase and lead to truncated cDNA molecules. This was combined with deep-sequencing in a developed method called ICE-seq. Computational methods for automated analysis of the data are available, the main

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The existence of two A-to-I modifications in relatively close proximity, which is common in Alu elements, means the downstream mod is less likely to be detected since the cDNA synthesis will be truncated at a prior nucleotide. The throughput is low, and the initial method required specific primers;

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is introduced to the cells so that it could be incorporated into nascent RNA in place of cytosine. Normally, the methyltransferases are released (i.e. covalent bond between cytosine and methyltransferase is broken) following methylation of the residue. For 5-aza-C, due to a nitrogen substitution in

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A modified version of bisulfite sequencing was developed by Schaefer et al. (2009) which decreased the temperature at which bisulfite treatment of RNA from 95 °C to 60 °C. The rationale behind the modification was that since RNA, unlike DNA, is not double-stranded, but rather, consists of

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m6A-CLIP (crosslinking immunoprecipitation) and miCLIP (m6A individual-nucleotide-resolution crosslinking and immunoprecipitation) are UV-based sequencing techniques. These two methods activate crosslinking at 254 nm, fragments RNA molecules before immunoprecipitation with antibody, and do not

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When the first nucleotide of a transcript is an adenosine, in addition to the ribose 2’-O-methylation, this base can be further methylated at the N6 position. m6A-seq was confirmed to be able to detect m6Am peaks at transcription start sites. Adapter ligation at both ends of RNA fragment results in

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The two methods were optimized to detect methylation peaks in poly(A)+ mRNA, but the protocol could be adapted to profile any type of RNA. Collected RNA sample is fragmented into ~100-nucleotide-long oligonucleotides using a fragmentation buffer, immunoprecipitation with purified anti-m6A antibody,

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The first method to detect A-to-I RNA modifications, developed in 1997, was inosine-specific cleavage. RNA samples are treated with glyoxal and borate to specifically modify all G bases, and subsequently enzymatically digested to by RNase T1, which cleaves after I sites. The amplification of these

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and the m5C-RNA methyltransferase of interest is immunoprecipitated along with the RNA molecules that are covalently linked to the protein. The IP step enabled >200-fold enrichment of RNA targets, which were mainly tRNAs. The enriched molecules were then fragmented and purified. cDNA library is

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are added to the eluate and supernatant, as well as an independent control arm consisting of just ERCC spike in. After antibody cleavage in the eluate pool, each of the three mixtures are sequenced on a next generation sequencing platform. The m6A levels per site or gene could be quantified by the

826:(SAM) --> methyl group on RNA base, the labelling of dietary methionine with C and D means these will end up in hm5C residues that have been altered since the addition of these into the diet. In contrast to m5C, a large quantity of hm5C modifications have been recorded within coding sequences. 734:

events in snRNAs. Pseudouridine has one more hydrogen bond donor from an imino group and a more stable C–C bond, since a C-glycosidic linkage has replaced the N-glycosidic linkage found in its counterpart (regular uridine). As neither of these changes affect its base-pairing properties, both will

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A novel method, Nm-REP-seq, was developed for the transcriptome-wide identification of 2'-O-methylation sites at single-base resolution by using RNA exoribonuclease (Mycoplasma genitalium RNase R, MgR) and periodate oxidation reactivity to eliminate 2'-hydroxylated (2'-OH) nucleosides. Nm-REP-seq

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There are multiple pseudouridine detection methods beginning with the addition of N-cyclohexyl-N′-b-(4-methylmorpholinium) ethylcarbodiimide metho-p-toluene-sulfonate (CMCT; also known as CMC), since its reaction with pseudouridine produces CMC-Ψ. CMC-Ψ causes reverse transcriptase to stall one

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Since the chemical makeup of inosine is a deaminated adenosine, this is one of few methylation alterations that has an accompanying alteration in base pairing, which can be capitalised on. The original adenosine nucleotide will pair with a thymine, whereas the methylated inosine will pair with a

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SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography) is used determining the fraction of RNA in a sample that carries a methylated adenine at a specific site. One can start with total RNA without having to enrich for the

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between crosslinking site and antibody. Peptide fragments that remain after antibody removal from RNA cause the base to be read as a C as opposed to a T during reverse transcription, effectively inducing a point mutation at the 4SU crosslinking site. The short fragments are subjected to library

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The resolution of these methods is 100-200nt, which was the range of the fragment size. These two methods had several drawbacks: (1) required substantial input material, (2) low resolution which made pinpointing the actual site with the m6A mark difficult, and (3) cannot directly assess false

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is introduced to the sample to digest all RNA, except for the RNA molecules with the 116-mers DNA attached. This radiolabeled product is then isolated and digested by nuclease to generate a mixture of modified and unmodified adenosines (5’P-m6A and 5’-P-A) which is separated using thin layer

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In m6A-CLIP and miCLIP, RNA is fragmented to ~20-80nt first, then the 254 nm UV-induced covalent RNA/m6A antibody complex was formed in the fragments containing m6A. The antibody was removed with proteinase K before reverse-transcription, library construction and sequencing. Remnants of

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In 2013, a modified version of m6A-seq based on the previous two methods m6A-seq and MeRIP-seq came out which aimed to increase resolution, and demonstrated this in the yeast transcriptome. They achieved this by decreasing fragment size and employing a ligation-based strand-specific library

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The modification of A to I is effected by adenosine deaminases that act on RNA (ADARs), of which in mice there are three. The knockdown of these in the cell, therefore, and the subsequent cell–cell comparison of ADAR+ and ADAR- RNA content would be anticipated to provide a basis for A-to-I

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Especially in MeRIP-Seq, the bioinformatics tools that are currently available are only able to call 1 site per ~100-200nt wide peak, so a substantial portion of clustered m6As (~64nt between each individual site within a cluster) are missed. Each cluster can contain up to 15 m6A residues.

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and to have roles in regulation. While 5-hydroxymethylcytidine (hm5dC) is known to be found in DNA in a widespread manner, hm5C is also found in organisms for which no hm5dC has been detected, indicating it is a separate process with distinct regulatory stipulations. To observe the

203:, some incorporation sites presumably near m6A location. Immunoprecipitation is then performed on full-length RNA using m6A-specific antibody . UV light at 365 nm is then shined onto RNA to activate the crosslinking to the antibody with 4SU. Crosslinked RNA was isolated via 1622:

Zhang, Ping; Huang, Junhong; Zheng, Wujian; Chen, Lifan; Liu, Shurong; Liu, Anrui; Ye, Jiayi; Zhou, Jie; Chen, Zhirong; Huang, Qiaojuan; Liu, Shun; Zhou, Keren; Qu, Lianghu; Li, Bin; Yang, Jianhua (28 October 2022). "Single-base resolution mapping of 2′-O-methylation sites by an

606: 555:. This ring opening results in preferential pairing with cytosine and is therefore read as guanosine during sequencing. This C to G transversion allows for base resolution detection of m5C sites. One caveat is that m5C sites not replaced by 5-azacytosine will be missed. 250:, which has higher local resolution around a specific site, (see below), implicating m6A-CLIP and miCLIP has high spatial resolution and low false discovery rate. miCLIP has been used to detect m6Am by looking at crosslinking-induced truncation sites at the 5’UTR. 461:

would be unchanged by the treatment. Previous attempts to develop m5C sequencing protocols using bisulfite treatment were not able to effectively address the problem of the harsh treatment of RNA which causes significant degradation of the molecules. Specifically,

91: 159:), are also the first methods to allow for any type of RNA modification sequencing. These methods were able to detect 10,000 m6A peaks in the mammalian transcriptome; the peaks were found to be enriched in 3’UTR regions, near STOP codons, and within long exons. 430:), similar to m6C sequencing. The second is detecting targets of m5C RNA methyltransferases by covalently linking the enzyme to its target, and then using IP specific to the target enzyme to enrich for RNA molecules containing the mark (Aza-IP and miCLIP). 52: 328:

ERCC-normalized RNA abundances in different pools. Since full-length RNA is used, it is possible to directly compare alternatively spliced isoforms between the m6A+ and m6A- fractions as well as comparing isoform abundance within the m6A+ portion.

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Although m6A sites could be profiled at high resolution using UV-based methods, the stoichiometry of m6A sites - the methylation status or the ratio m6A+ to m6A- for each individual site within a type of RNA - is still unknown. SCARLET (2013) and

1000:(RNN) trained with known sequences, it was possible to demonstrate that the modified nucleotides produce a characteristic disruption in the ionic current when passing through the pore, and that these data can be used to identify the nucleotide. 401: 331:

Despite the advances in m6A-sequencing, several challenges still remain: (1) A method has yet to be developed that characterizes the stoichiometry between different sites in the same transcript; (2) Analysis results are heavily dependent on the

139: 777:. One final limitation is that, for CMC labelling of pseudouridine to be specific, it is not complete, and therefore nor is it quantitative. A new reactant that could achieve a higher sensitivity with specificity would be beneficial. 87:. Various sequencing methods have been developed to profile each type of modification. The scale, resolution, sensitivity, and limitations associated with each method and the corresponding bioinformatics tools used will be discussed. 1572:

Boccaletto, P; Stefaniak, F; Ray, A; Cappannini, A; Mukherjee, S; Purta, E; Kurkowska, M; Shirvanizadeh, N; Destefanis, E; Groza, P; Avşar, G; Romitelli, A; Pir, P; Dassi, E; Conticello, SG; Aguilo, F; Bujnicki, JM (7 January 2022).

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premise being the comparison of treated and untreated samples to identify truncated transcripts and thus infer an inosine modification by read count, with a step to reduce false positives by comparison to online database dbSNP.

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target RNA molecule. Therefore, it is an especially suitable method for quantifying methylation status in low abundance RNAs such as tRNAs. However, it is not suitable or practical for large-scale location of m6A sites.

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regions of single-strandedness, double-stranded stem structures and loops, it could be possible to unwind RNA at a much lower temperature. Indeed, RNA could be treated for 180 minutes at 60C without significant loss of

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Bakin, A.; Ofengand, J. (1993). "Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique".

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fragments then allows analysis of cleavage sites and inference of A-to-I modification. . It was used to prove the position of inosine at specific sites rather than identify novel sites or transcriptome-wide profiles.

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hMeRIP-seq is an immunoprecipitation method, in which RNA–protein complexes are crosslinked for stability, and antibodies specific to hm5C are added. Using this method, over 3,000 hm5C peaks have been called in

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to cDNA, especially at the +1 position to the m6A site (5’ to the m6A site) in the sequence reads. Positive sites seen using m6A-CLIP and miCLIP had high percent of matches with those detected using

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such as tRNA. An induced mutation of C271A in NSUN2 inhibits release of enzyme from RNA target. This mutation was over-expressed in the cells of interest, and the mutated NSUN2 was also tagged with the

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The background noise caused by single nucleotide polymorphisms (SNPs), somatic mutations, pseudogenes and sequencing errors reduce the reliability of the signal, especially in a single-cell context.

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Sakurai, Masayuki; Yano, Takanori; Kawabata, Hitomi; Ueda, Hiroki; Suzuki, Tsutomu (2010). "Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome".

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used to call the peaks; (3) Current methods all use m6A-specific antibodies to tag m6A sites, but it has been reported that the antibodies contain intrinsic bias for RNA sequences.

884:(two-dimensional thin-layer chromatography). This method is able to detect and quantify every modification but will not contribute to the characterization of the sequence. 937:, that exploits the decrease in duplex stability of cDNA oligonucleotides, due to the impediment in conventional base-pairing caused by modifications (ex. m1A, m1G, m22G) 2107:

Suzuki, Tsutomu; Ueda, Hiroki; Okada, Shunpei; Sakurai, Masayuki (2015). "Transcriptome-wide identification of adenosine-to-inosine editing using the ICE-seq method".

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Both miCLIP and Aza-IP, though limited by specific targeting of enzymes, can allow for the detection of low-abundance methylated RNA without deep sequencing.

580:. The covalently linked RNA-protein complexes are isolated via immunoprecipitation for a Myc-specific antibody. These complexes are confirmed and detected by 319:

added to the mixture to ensure all m6A-containing RNAs are pulled down. The mixture is separated into eluate (m6A+ RNAs) and supernatant (m6A- RNAs) pools.

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Huber, Sabrina M.; van Delft, Pieter; Mendil, Lee; Bachman, Martin; Smollett, Katherine; Werner, Finn; Miska, Eric A.; Balasubramanian, Shankar (2015).

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or according to the SCARLET method. In both cases, the final product is a group of 5’ nucleoside monophosphates (5’ NMPs) that will be analyzed by TLC.

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nucleotide in the 3’ direction. These methods have single-nucleotide resolution. In an optimisation step, azido-CMC can confer the ability to add

722:, this can occur by either of two distinct mechanisms; it is sometimes referred to as the ‘fifth RNA nucleotide’. It is incorporated into stable 789:

once for 5-hydroxylmethylcytidine (hm5C), or oxidised twice for 5-formylcytidine (f5C). Arising from the oxidative processing of m5C enacted in

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Licht, Konstantin; Kapoor, Utkarsh; Amman, Fabian; Picardi, Ernesto; Martin, David; Bajad, Prajakta; Jantsch, Michael F. (September 2019).

952: 535:-tagged m5C-RNA methytransferase derivative so that the antibody used later on for immunoprecipitation could recognize the enzyme. Second, 564: 2438:"Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy" 270:

within which m6A sites are usually found, and take into consideration all possible motifs. Therefore, it is less likely to miss sites.

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was added on top of m6A-seq to produce PA-m6A-seq (photo-crosslinking-assisted m6A-seq) which increases resolution up to ~23nt. First,

588:. The RNA is then extracted from the complex, reverse-transcribed, amplified with PCR, and sequenced using next-generation platforms. 540:

the C5 position of cytosine, the RNA methytransferase enzyme remains covalently bound to the target RNA molecule at the C6 position.

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Despite two distinct base-resolution methods being available for hm5dC, there are no base-resolution methods for detection of hm5C.

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Two principles have been used to develop m5C sequencing methods. The first one is antibody-based approach (bisuphite sequencing and

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methods. This modification is marked by the methylation of the adenosine base at the nitrogen-6 position. It is abundantly found in

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to cleave the RNA strand precisely at the 5’-end of the candidate site. The cut site is then radiolabeled with phosphorus-32 and

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has 2’OMe/2’H modifications and is complementary to the target sequence. The chimeric oligonucleotide serves as a guide to allow

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Shafik, A.; Schumann, U.; Evers, M.; Sibbritt, T.; Preiss, T. (2016). "The emerging epitranscriptomics of long noncoding RNAs".

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A possible alternative to the detection of epitranscriptomic modifications by SMRT sequencing is the direct detection using the

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design. The method causes a lot of RNA degradation, so it is necessary to start with a large amount of sample, or use effective

715: 753:; subsequent biotin pulldown will enrich Ψ-containing transcripts, allowing identification of even low-abundance transcripts. 551:

An important additional feature is that RNA methyltransferase covalent linkage to the C5 of m-aza-C induces rearrangement and

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analysis. Since the metabolic pathway from nutritional intake to nucleotide incorporation is known to progress from dietary

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Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V. (2014).

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have the same output when directly sequenced; therefore methods for its detection involve prior biochemical modification.

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Saletore, Yogesh; Meyer, Kate; Korlach, Jonas; Vilfan, Igor D.; Jaffrey, Samie; Mason, Christopher E. (October 2012).

416: 320: 2389:"rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells" 411:, m5C, is abundantly found in mRNA and ncRNAs, especially tRNA and rRNAs. In tRNAs, this modification stabilizes the 155:-wide profile of m6A in mammalian cells. These two techniques, called m6A-seq and MeRIP-seq (m6A-specific methylated 2789: 898:

ligated DNA oligonucleotide and finally hydrolyzed and therefore released thanks to the activity of the Nuclease P1.

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ICE-based and CMC-based detection of inosine and pseudouridine. In both cases a truncated cDNA molecule is produced.

2928: 1476:"Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA" 766: 762: 470:. As a result, it is difficult to pre-enrich RNA molecules or to obtain enough PCR product of the correct size for 362: 120: 20:, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the 497: 997: 770: 385: 333: 263:(2016) allows for the quantitation of stoichiometry at a specific locus and transcriptome-wide, respectively. 2152:"A high resolution A-to-I editing map in the mouse identifies editing events controlled by pre-mRNA splicing" 2923: 1247:"High-Resolution Mapping Reveals a Conserved, Widespread, Dynamic mRNA Methylation Program in Yeast Meiosis" 837: 420: 156: 76: 2859: 1174:

Dominissini; et al. (2012). "Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq".

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As with other procedures predicated on biochemical alteration followed by sequencing, the development of

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chromatography. The relative proportions of the two groups can be determined using UV absorption levels.

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Morse, D. P.; Bass, B. L. (1997). "Detection of inosine in messenger RNA by inosine-specific cleavage".

242: 1435:"N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases" 2858:

Garalde, DR; Snell, EA; Jachimowicz, D; Sipos, B; Lloyd, JH; Bruce, M; Pantic, N; et al. (2018).

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Basturea, Georgeta N. (2013). "Research Methods for Detection and Quantitation of RNA Modifications".

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Morse, D.P.; Bass, B.L. (1997). "Detection of inosine in messenger RNA by inosine-specific cleavage".

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Perturbation of m6A Writers Reveals Two Distinct Classes of mRNA Methylation at Internal and 50 Sites

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Method of inosine identification: Inosine base pairs with cytidine while adenosine pairs with uracil.

388:, the methods used for m6A profiling can be and were adapted for m6Am profiling, namely m6A-seq, and 238: 33: 28:

analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for

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at the crosslinking site on the RNA after antibody removal, leads to insertions, truncations, and

2890: 2238: 2132: 1648: 1207: 985: 931:, that exploits the ligase sensitivity to 3’ and 5’ nucleotides (so far used for m6A, 2’-O-Me, Ψ) 891: 577: 513: 116: 60: 1315:"A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation" 306: 2882: 2826: 2790:"The birth of the Epitranscriptome: deciphering the function of RNA modifications / Full Text" 2770: 2726: 2677: 2636: 2600: 2516: 2467: 2418: 2369: 2320: 2279: 2230: 2189: 2171: 2124: 2089: 2051: 2013: 1978: 1927: 1880: 1857: 1812: 1761: 1704: 1640: 1604: 1554: 1505: 1456: 1412: 1344: 1276: 1199: 1151: 1122:"Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3'UTRs and near Stop Codons" 1095: 1077: 858: 815: 798: 486: 324: 2749:"RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine" 536: 485:

After bisulfite treatment of fragmented RNA, reverse transcription is performed, followed by

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Bioinformatics methods used to analyze m6A peaks do not make any prior assumptions about the

247: 2874: 2816: 2806: 2760: 2716: 2708: 2667: 2628: 2590: 2582: 2543: 2506: 2498: 2457: 2449: 2408: 2400: 2359: 2351: 2310: 2269: 2220: 2179: 2163: 2116: 2081: 2043: 2005: 1968: 1958: 1917: 1909: 1847: 1839: 1802: 1792: 1751: 1743: 1694: 1686: 1632: 1594: 1586: 1544: 1536: 1495: 1487: 1446: 1402: 1394: 1334: 1326: 1266: 1258: 1191: 1141: 1133: 1085: 1069: 989: 881: 408: 389: 370: 72: 37: 2563:"Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells" 785:

Cytidine residues, modified once to m5C (discussed above), can be further modified: either

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ligand binding and translational fidelity in tRNA, and in fine-tuning branching events and

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of the expected size. Deamination rates were determined to be 99% at 180min of treatment.

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Identification of direct targets and modified bases of RNA cytosine methyltransferases

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addition of methyl groups to cytosine RNA residues followed by oxidative processing,

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Modified bisulfite sequencing was optimized for rRNA, tRNA, and miRNA molecules from

212: 152: 84: 2242: 90: 2913: 2894: 2315: 2298: 2136: 1383:"Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome" 1211: 904:

This method has proven to be very useful in the validation of modified residues in

731: 552: 283: 208: 200: 1947:"Widespread A-to-I RNA Editing of Alu-Containing mRNAs in the Human Transcriptome" 2404: 2274: 2257: 1963: 1843: 1058:"Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications" 289:

annealing to the target RNA around the candidate modification site. The chimeric

1898:"A biochemical landscape of A-to-I RNA editing in the human brain transcriptome" 956: 528: 59:

There are seven major classes of chemical modifications found in RNA molecules:

51: 1262: 1137: 765:

has removed the limitations requiring prior knowledge of sites of interest and

2811: 2502: 1636: 819: 442: 302: 2175: 1081: 2765: 2748: 2225: 2208: 1747: 1525:"m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome" 719: 490: 400: 108: 104: 2886: 2830: 2774: 2730: 2712: 2681: 2604: 2520: 2471: 2422: 2373: 2324: 2283: 2234: 2193: 2128: 2120: 2055: 1982: 1931: 1861: 1816: 1765: 1708: 1644: 1608: 1590: 1558: 1509: 1491: 1460: 1416: 1348: 1330: 1280: 1203: 1155: 1099: 496:

Since the developers of the method used the Roche platform, they also used

2672: 2655: 2640: 2167: 2093: 2017: 1913: 1073: 955:(SMRT) is used in the epigenomic and epitranscriptomic fields. As regards 2547: 2453: 2047: 1690: 786: 727: 450: 2632: 2586: 1195: 151:

In 2012, the first two methods for m6A sequencing came out that enabled

138: 2878: 1852: 1540: 1451: 1434: 1398: 909: 811: 599: 532: 454: 366: 294: 234: 80: 2085: 2009: 1779:

Hussain, S.; Aleksic, J.; Blanco, S.; Dietmann, S.; Frye, M. (2013).

1044:

Epitranscriptomic Sequencing Technologies: decoding RNA modifications

790: 750: 489:

of the cDNA products, and finally deep sequencing was done using the

112: 2355: 948:

Single-Molecule Real-Time Sequencing for epitranscriptome sequencing

1797: 1781:"Characterizing 5-methylcytosine in the mammalian epitranscriptome" 905: 568: 544: 521: 517: 290: 132: 128: 124: 41: 524:— the two main enzymes responsible for laying down the m5C mark. 2860:"Highly parallel direct RNA sequencing on an array of nanopores" 1575:"MODOMICS: a database of RNA modification pathways. 2021 update" 807: 602:

is created enzymatically when an adenosine residue is modified.

445:. Bisulfite treatment has been most widely used to detect dm5C ( 45: 29: 1724:"Messenger RNA modifications: form, distribution, and function" 865:, other two validation techniques have been developed, namely 1376: 1374: 1372: 1370: 1368: 1366: 1364: 1362: 1360: 1358: 913: 794: 466:

treatment (high pH) of RNA is detrimental to the stability of

1945:

Athanasiadis, Alekos; Rich, Alexander; Maas, Stefan (2004).

1675:"RNA cytosine methylation analysis by bisulphite sequencing" 55:

Schematic diagram of epitranscriptomic sequencing workflows.

2697:"Formation and Abundance of 5-Hydroxymethylcytosine in RNA" 663:

Inosine chemical erasing (ICE) refer to a process in which

935:

Microarray modification identification through a DNA-chip

2029: 2027: 512:

has been optimized on and used for detecting targets of

2340:"Transcriptome-wide dynamics of RNA pseudouridylation" 797:) family enzymes, hm5C is known to occur in all three 358:

Methods for N6,2'-O-dimethyladenosine (m6Am) Profiling

1056:

Licht, Konstantin; Jantsch, Michael F. (2016-04-11).

119:(mA) cannot be detected using standard sequencing or 565:

Methylation induced crosslinking immunoprecipitation

199:(4SU) is incorporated into the RNA by adding 4SU in 718:, is created when a uridine base is isomerised. In 357: 1038: 1036: 1034: 1032: 655:the protocol is complicated and labour-intensive. 2209:"Pseudouridine in RNA: what, where, how, and why" 1875: 1873: 1871: 1722:Gilbert, W.V.; Bell, T.A.; Schaening, C. (2016). 1240: 1238: 1236: 1234: 1030: 1028: 1026: 1024: 1022: 1020: 1018: 1016: 1014: 1012: 2256:Kiss, T.; Fayet-Lebaron, E.; Jady, B.E. (2010). 1428: 1426: 373:, when an additional methyl group is added to a 369:mRNAs, occurs at the first nucleotide after the 1115: 1113: 1111: 1109: 699:Methods for Pseudouridine Methylation Profiling 254:Methods for quantifying m6A modification status 142:Methods developed to profile N-methyladenosine. 941:RT primer extension at low dNTPs concentration 810:can be fed on a diet incorporating particular 781:Methods for 5-hydroxylmethylcytidine Profiling 548:then constructed and sequencing is performed. 510:5-azacytidine-mediated RNA immunoprecipitation 404:Methods developed to profile 5-methylcytidine. 107:does not affect its ability to base-pair with 2067: 2065: 1891: 1889: 929:Splinted ligation of particular modified DNAs 726:such as tRNA, rRNA, and snRNA, with roles in 392:(see m6A-seq and miCLIP descriptions above). 8: 2654:Kellner, S.; Burhenne, J.; Helm, M. (2010). 1623:exoribonuclease-enriched chemical method". 853:Biophysical validation of RNA modifications 1308: 1306: 1304: 1302: 1300: 1298: 1296: 1294: 1292: 1290: 1169: 1167: 1165: 2820: 2810: 2764: 2720: 2671: 2594: 2510: 2461: 2436:Baudin-Baillieu, A.; et al. (2009). 2412: 2363: 2338:Karijolich, J.; Yi, C.; Yu, Y.T. (2015). 2314: 2273: 2224: 2183: 1972: 1962: 1921: 1851: 1806: 1796: 1755: 1698: 1598: 1548: 1499: 1450: 1406: 1338: 1270: 1145: 1089: 975:Nanopore sequencing in epitranscriptomics 2297:Hamma, T.; Ferre-D; Amare, A.R. (2006). 1046:. Nature Methods. doi:10.1038/NMETH_4110 702: 604: 449:). The treatment essentially converts a 399: 377:residue at the ‘capped’ 5ʹ end of mRNA. 137: 89: 50: 2487:"Pseudouridines in spliceosomal snRNAs" 1883:. Nature Biotechnology. 31(5): 459-464. 1008: 923:: this method includes several variants 571:targets, which were found to be mostly 301:to a 116nt ssDNA oligonucleotide using 99:Methods for profiling N-methyladenosine 94:The major classes of RNA modifications. 1879:Khoddami, V. and Cairns, B.R. (2013). 1668: 1666: 1664: 1662: 676:The original ICE protocol involved an 396:Methods for 5-methylcytidine profiling 340:Methods for 2'-O-methylation Profiling 339: 2742: 2740: 1042:Li, X, Xiong, X., and Yi, C. (2017). 619:cytosine. cDNA sequences obtained by 7: 2258:"Box H/ACA small ribonucleoproteins" 953:Single-molecule real-time sequencing 207:and fragmented further to ~25-30nt; 193:UV-induced RNA-antibody crosslinking 2485:Yu, A.T.; Ge, J.; Yu, Y.T. (2011). 943:, for mapping of RT arrest signals. 716:post-translational RNA modification 614:Analysis of base-pairing properties 1673:Schaefer, M.; et al. (2008). 1245:Schwartz, S.; et al. (2013). 870:Pre- and post-labelling techniques 714:, or Ψ, the overall most abundant 127:+ mRNA; also found in tRNA, rRNA, 14: 2747:Delatte, B.; et al. (2016). 2207:Charette, M.; Gray, M.W. (2000). 1896:Sakurai, M.; et al. (2014). 1523:Molinie, B.; et al. (2016). 1120:Meyer, K.D.; et al. (2012). 2656:"Detection of RNA modifications" 1381:Linder, B.; et al. (2015). 921:Oligonucleotide-based techniques 417:anticodon stem-loop conformation 380:Since m6Am can be recognized by 321:External RNA Controls Consortium 24:, or (2) improving or modifying 2840:from the original on 2022-10-17 963:(ZMWs) are used to capture the 32:molecules, except for modified 2387:Jack, K.; et al. (2011). 2316:10.1016/j.chembiol.2006.09.009 1: 793:by ten-eleven translocation ( 595:Methods for Inosine Profiling 437:Modified bisulfite sequencing 2405:10.1016/j.molcel.2011.09.017 2275:10.1016/j.molcel.2010.01.032 1964:10.1371/journal.pbio.0020391 1844:10.1016/j.bbagrm.2015.10.019 1313:Ke, S.; et al. (2015). 1224:Schwartz, S. et al. (2014). 498:GS Amplicon Variant Analyzer 282:The procedure begins with a 18:epitranscriptomic sequencing 1625:Science China Life Sciences 1433:Maity, A.; Das, B. (2016). 1228:. Cell Reports. 8: 284-296. 527:First, the cell is made to 211:was used to dissociate the 2945: 1263:10.1016/j.cell.2013.10.047 1138:10.1016/j.cell.2012.05.003 814:and these be traced by LC- 773:techniques to account for 763:high-throughput sequencing 2812:10.1186/gb-2012-13-10-175 2503:10.1007/s13238-011-1087-1 2299:"Pseudouridine synthases" 1637:10.1007/s11427-022-2210-0 1474:Liu; et al. (2013). 998:recurrent neural networks 641:Inosine-specific cleavage 386:transcription start sites 363:N6,2'-O-dimethyladenosine 69:N6,2'-O-dimethyladenosine 2344:Nat. Rev. Mol. Cell Biol 419:. In rRNAs, m5C affects 334:bioinformatics algorithm 77:5-hydroxylmethylcytidine 40:which was optimized for 2766:10.1126/science.aac5253 2226:10.1080/152165400410182 2036:Nature Chemical Biology 1748:10.1126/science.aad8711 1319:Genes & Development 1062:Journal of Cell Biology 838:Drosophila melanogaster 157:RNA immunoprecipitation 2713:10.1002/cbic.201500013 2121:10.1038/nprot.2015.037 1832:Biochim. Biophys. Acta 1679:Nucleic Acids Research 1579:Nucleic Acids Research 1492:10.1261/rna.041178.113 1331:10.1101/gad.269415.115 708: 610: 421:translational fidelity 405: 143: 95: 56: 2673:10.4161/rna.7.2.11468 2536:Materials and Methods 2168:10.1101/gr.242636.118 1914:10.1101/gr.162537.113 1074:10.1083/jcb.201511041 706: 608: 567:) was used to detect 464:bisulfite deamination 403: 243:reverse transcription 147:m6A-seq and MeRIP-seq 141: 93: 54: 2548:10.13070/mm.en.3.186 2048:10.1038/nchembio.434 1591:10.1093/nar/gkab1083 994:Hidden Markov Models 961:zero-mode waveguides 824:S-adenosylmethionine 775:amplification biases 678:RT-PCR amplification 468:phosphodiester bonds 459:methylated cytosines 375:2ʹ-O-methyladenosine 34:bisulfite sequencing 2805:(10). article 175. 2633:10.1021/bi00088a030 2587:10.1038/nature13802 2579:2014Natur.515..143C 1740:2016Sci...352.1408G 1734:(6292): 1408–1412. 1196:10.1038/nature11112 1188:2012Natur.485..201D 981:Nanopore sequencing 969:Pacific Biosciences 739:Biochemical methods 543:Third, the cell is 413:secondary structure 382:anti-m6A antibodies 224:m6A-CLIP and miCLIP 218:Illumina sequencing 205:competition elution 2879:10.1038/nmeth.4577 2454:10.1093/nar/gkp816 1691:10.1093/nar/gkn954 1541:10.1038/nmeth.3898 1452:10.1111/febs.13614 1399:10.1038/nmeth.3453 709: 685:Biological methods 611: 514:methyltransferases 406: 144: 96: 57: 2929:Molecular biology 2759:(6270): 282–285. 2627:(37): 9754–9762. 2573:(7525): 143–146. 2448:(22): 7665–7677. 2442:Nucleic Acids Res 2309:(11): 1125–1135. 2086:10.1021/bi9709607 2080:(28): 8429–8434. 2010:10.1021/bi9709607 2004:(28): 8429–8434. 1585:(D1): D231–D235. 1486:(12): 1848–1856. 1325:(19): 2037–2053. 1182:(7397): 201–208. 986:N-methyladenosine 859:mass spectrometry 487:PCR amplification 216:construction and 117:N-methyladenosine 61:N-methyladenosine 2936: 2899: 2898: 2864: 2855: 2849: 2848: 2846: 2845: 2839: 2824: 2814: 2794: 2785: 2779: 2778: 2768: 2744: 2735: 2734: 2724: 2692: 2686: 2685: 2675: 2651: 2645: 2644: 2615: 2609: 2608: 2598: 2558: 2552: 2551: 2531: 2525: 2524: 2514: 2482: 2476: 2475: 2465: 2433: 2427: 2426: 2416: 2384: 2378: 2377: 2367: 2335: 2329: 2328: 2318: 2294: 2288: 2287: 2277: 2253: 2247: 2246: 2228: 2204: 2198: 2197: 2187: 2162:(9): 1453–1463. 2147: 2141: 2140: 2109:Nature Protocols 2104: 2098: 2097: 2069: 2060: 2059: 2031: 2022: 2021: 1993: 1987: 1986: 1976: 1966: 1942: 1936: 1935: 1925: 1893: 1884: 1877: 1866: 1865: 1855: 1827: 1821: 1820: 1810: 1800: 1776: 1770: 1769: 1759: 1719: 1713: 1712: 1702: 1670: 1657: 1656: 1619: 1613: 1612: 1602: 1569: 1563: 1562: 1552: 1520: 1514: 1513: 1503: 1471: 1465: 1464: 1454: 1445:(9): 1607–1630. 1430: 1421: 1420: 1410: 1378: 1353: 1352: 1342: 1310: 1285: 1284: 1274: 1257:(6): 1409–1421. 1242: 1229: 1222: 1216: 1215: 1171: 1160: 1159: 1149: 1132:(7): 1635–1646. 1117: 1104: 1103: 1093: 1053: 1047: 1040: 990:5-methylcytosine 636:Chemical methods 409:5-methylcytidine 239:C to T mutations 183:UV-based Methods 73:5-methylcytidine 65:2'-O-methylation 38:5-methylcytidine 2944: 2943: 2939: 2938: 2937: 2935: 2934: 2933: 2904: 2903: 2902: 2862: 2857: 2856: 2852: 2843: 2841: 2837: 2792: 2787: 2786: 2782: 2746: 2745: 2738: 2694: 2693: 2689: 2653: 2652: 2648: 2617: 2616: 2612: 2560: 2559: 2555: 2533: 2532: 2528: 2484: 2483: 2479: 2435: 2434: 2430: 2386: 2385: 2381: 2356:10.1038/nrm4040 2350:(10): 581–585. 2337: 2336: 2332: 2296: 2295: 2291: 2255: 2254: 2250: 2206: 2205: 2201: 2156:Genome Research 2149: 2148: 2144: 2106: 2105: 2101: 2071: 2070: 2063: 2042:(10): 733–740. 2033: 2032: 2025: 1995: 1994: 1990: 1944: 1943: 1939: 1895: 1894: 1887: 1878: 1869: 1829: 1828: 1824: 1778: 1777: 1773: 1721: 1720: 1716: 1672: 1671: 1660: 1621: 1620: 1616: 1571: 1570: 1566: 1522: 1521: 1517: 1473: 1472: 1468: 1432: 1431: 1424: 1380: 1379: 1356: 1312: 1311: 1288: 1244: 1243: 1232: 1223: 1219: 1173: 1172: 1163: 1119: 1118: 1107: 1055: 1054: 1050: 1041: 1010: 1006: 977: 959:, thousands of 950: 855: 847: 832: 783: 759: 746: 741: 724:non-coding RNAs 701: 692: 687: 674: 661: 659:ICE and ICE-seq 652: 643: 638: 630: 616: 597: 573:non-coding RNAs 561: 516:, particularly 506: 472:deep sequencing 439: 433: 415:and influences 398: 360: 351: 342: 316: 287:oligonucleotide 276: 268:sequence motifs 256: 226: 190: 185: 149: 103:Methylation of 101: 12: 11: 5: 2942: 2940: 2932: 2931: 2926: 2924:Bioinformatics 2921: 2916: 2906: 2905: 2901: 2900: 2873:(3): 201–206. 2867:Nature Methods 2850: 2798:Genome Biology 2780: 2736: 2707:(5): 752–755. 2687: 2666:(2): 237–247. 2646: 2610: 2553: 2526: 2497:(9): 712–725. 2477: 2428: 2399:(4): 660–666. 2379: 2330: 2289: 2268:(5): 597–606. 2248: 2219:(5): 341–351. 2199: 2142: 2115:(5): 715–732. 2099: 2061: 2023: 1988: 1937: 1908:(3): 522–534. 1885: 1867: 1822: 1798:10.1186/gb4143 1771: 1714: 1658: 1631:(4): 800–818. 1614: 1564: 1535:(8): 692–698. 1529:Nature Methods 1515: 1466: 1422: 1393:(8): 767–772. 1387:Nature Methods 1354: 1286: 1230: 1217: 1161: 1105: 1048: 1007: 1005: 1002: 992:(5-mC). Using 976: 973: 965:DNA polymerase 949: 946: 945: 944: 938: 932: 925: 924: 902: 901: 900: 899: 888:Post-labelling 885: 874: 873: 863:chromatography 854: 851: 846: 843: 831: 828: 782: 779: 758: 755: 745: 742: 740: 737: 700: 697: 691: 690:ADAR knockdown 688: 686: 683: 673: 670: 660: 657: 651: 648: 642: 639: 637: 634: 629: 626: 615: 612: 596: 593: 560: 557: 505: 502: 438: 435: 397: 394: 365:, abundant in 359: 356: 350: 347: 341: 338: 315: 312: 299:splint-ligated 275: 272: 255: 252: 225: 222: 189: 186: 184: 181: 148: 145: 100: 97: 36:for profiling 26:bioinformatics 13: 10: 9: 6: 4: 3: 2: 2941: 2930: 2927: 2925: 2922: 2920: 2917: 2915: 2912: 2911: 2909: 2896: 2892: 2888: 2884: 2880: 2876: 2872: 2868: 2861: 2854: 2851: 2836: 2832: 2828: 2823: 2818: 2813: 2808: 2804: 2800: 2799: 2791: 2784: 2781: 2776: 2772: 2767: 2762: 2758: 2754: 2750: 2743: 2741: 2737: 2732: 2728: 2723: 2718: 2714: 2710: 2706: 2702: 2698: 2691: 2688: 2683: 2679: 2674: 2669: 2665: 2661: 2657: 2650: 2647: 2642: 2638: 2634: 2630: 2626: 2622: 2614: 2611: 2606: 2602: 2597: 2592: 2588: 2584: 2580: 2576: 2572: 2568: 2564: 2557: 2554: 2549: 2545: 2541: 2537: 2530: 2527: 2522: 2518: 2513: 2508: 2504: 2500: 2496: 2492: 2488: 2481: 2478: 2473: 2469: 2464: 2459: 2455: 2451: 2447: 2443: 2439: 2432: 2429: 2424: 2420: 2415: 2410: 2406: 2402: 2398: 2394: 2390: 2383: 2380: 2375: 2371: 2366: 2361: 2357: 2353: 2349: 2345: 2341: 2334: 2331: 2326: 2322: 2317: 2312: 2308: 2304: 2300: 2293: 2290: 2285: 2281: 2276: 2271: 2267: 2263: 2259: 2252: 2249: 2244: 2240: 2236: 2232: 2227: 2222: 2218: 2214: 2210: 2203: 2200: 2195: 2191: 2186: 2181: 2177: 2173: 2169: 2165: 2161: 2157: 2153: 2146: 2143: 2138: 2134: 2130: 2126: 2122: 2118: 2114: 2110: 2103: 2100: 2095: 2091: 2087: 2083: 2079: 2075: 2068: 2066: 2062: 2057: 2053: 2049: 2045: 2041: 2037: 2030: 2028: 2024: 2019: 2015: 2011: 2007: 2003: 1999: 1992: 1989: 1984: 1980: 1975: 1970: 1965: 1960: 1956: 1952: 1948: 1941: 1938: 1933: 1929: 1924: 1919: 1915: 1911: 1907: 1903: 1899: 1892: 1890: 1886: 1882: 1876: 1874: 1872: 1868: 1863: 1859: 1854: 1849: 1845: 1841: 1837: 1833: 1826: 1823: 1818: 1814: 1809: 1804: 1799: 1794: 1790: 1786: 1782: 1775: 1772: 1767: 1763: 1758: 1753: 1749: 1745: 1741: 1737: 1733: 1729: 1725: 1718: 1715: 1710: 1706: 1701: 1696: 1692: 1688: 1684: 1680: 1676: 1669: 1667: 1665: 1663: 1659: 1654: 1650: 1646: 1642: 1638: 1634: 1630: 1626: 1618: 1615: 1610: 1606: 1601: 1596: 1592: 1588: 1584: 1580: 1576: 1568: 1565: 1560: 1556: 1551: 1546: 1542: 1538: 1534: 1530: 1526: 1519: 1516: 1511: 1507: 1502: 1497: 1493: 1489: 1485: 1481: 1477: 1470: 1467: 1462: 1458: 1453: 1448: 1444: 1440: 1436: 1429: 1427: 1423: 1418: 1414: 1409: 1404: 1400: 1396: 1392: 1388: 1384: 1377: 1375: 1373: 1371: 1369: 1367: 1365: 1363: 1361: 1359: 1355: 1350: 1346: 1341: 1336: 1332: 1328: 1324: 1320: 1316: 1309: 1307: 1305: 1303: 1301: 1299: 1297: 1295: 1293: 1291: 1287: 1282: 1278: 1273: 1268: 1264: 1260: 1256: 1252: 1248: 1241: 1239: 1237: 1235: 1231: 1227: 1221: 1218: 1213: 1209: 1205: 1201: 1197: 1193: 1189: 1185: 1181: 1177: 1170: 1168: 1166: 1162: 1157: 1153: 1148: 1143: 1139: 1135: 1131: 1127: 1123: 1116: 1114: 1112: 1110: 1106: 1101: 1097: 1092: 1087: 1083: 1079: 1075: 1071: 1067: 1063: 1059: 1052: 1049: 1045: 1039: 1037: 1035: 1033: 1031: 1029: 1027: 1025: 1023: 1021: 1019: 1017: 1015: 1013: 1009: 1003: 1001: 999: 995: 991: 987: 982: 974: 972: 970: 966: 962: 958: 954: 947: 942: 939: 936: 933: 930: 927: 926: 922: 919: 918: 917: 915: 911: 907: 896: 895: 893: 889: 886: 883: 879: 878:Pre-labelling 876: 875: 871: 868: 867: 866: 864: 860: 852: 850: 844: 842: 840: 839: 829: 827: 825: 821: 817: 813: 809: 805: 800: 796: 792: 788: 780: 778: 776: 772: 771:normalisation 768: 764: 756: 754: 752: 751:biotinylation 743: 738: 736: 733: 729: 725: 721: 717: 713: 712:Pseudouridine 705: 698: 696: 689: 684: 682: 679: 671: 669: 666: 665:acrylonitrile 658: 656: 649: 647: 640: 635: 633: 627: 625: 622: 613: 607: 603: 601: 594: 592: 589: 587: 586:phosphorus-32 583: 582:radiolabeling 579: 574: 570: 566: 558: 556: 554: 549: 546: 541: 538: 534: 530: 525: 523: 519: 515: 511: 503: 501: 499: 494: 492: 488: 483: 481: 480:PCR amplicons 475: 473: 469: 465: 460: 456: 452: 448: 444: 436: 434: 431: 429: 424: 422: 418: 414: 410: 402: 395: 393: 391: 387: 383: 378: 376: 372: 368: 364: 355: 348: 346: 337: 335: 329: 326: 322: 313: 311: 308: 304: 300: 296: 292: 288: 285: 280: 273: 271: 269: 264: 262: 253: 251: 249: 244: 240: 236: 230: 223: 221: 219: 214: 213:covalent bond 210: 206: 202: 198: 197:4-thiourodine 194: 187: 182: 180: 176: 172: 168: 164: 160: 158: 154: 153:transcriptome 146: 140: 136: 134: 130: 126: 122: 121:hybridization 118: 114: 110: 106: 98: 92: 88: 86: 85:pseudouridine 82: 78: 74: 70: 66: 62: 53: 49: 47: 43: 39: 35: 31: 27: 23: 22:RNA sequencer 19: 2870: 2866: 2853: 2842:. Retrieved 2802: 2796: 2783: 2756: 2752: 2704: 2700: 2690: 2663: 2659: 2649: 2624: 2621:Biochemistry 2620: 2613: 2570: 2566: 2556: 2539: 2535: 2529: 2494: 2491:Protein Cell 2490: 2480: 2445: 2441: 2431: 2396: 2392: 2382: 2347: 2343: 2333: 2306: 2302: 2292: 2265: 2261: 2251: 2216: 2212: 2202: 2159: 2155: 2145: 2112: 2108: 2102: 2077: 2074:Biochemistry 2073: 2039: 2035: 2001: 1998:Biochemistry 1997: 1991: 1957:(12): e391. 1954: 1951:PLOS Biology 1950: 1940: 1905: 1901: 1838:(1): 59–70. 1835: 1831: 1825: 1788: 1784: 1774: 1731: 1727: 1717: 1682: 1678: 1628: 1624: 1617: 1582: 1578: 1567: 1532: 1528: 1518: 1483: 1479: 1469: 1442: 1439:FEBS Journal 1438: 1390: 1386: 1322: 1318: 1254: 1250: 1220: 1179: 1175: 1129: 1125: 1068:(1): 15–22. 1065: 1061: 1051: 978: 951: 940: 934: 928: 920: 903: 887: 877: 869: 856: 848: 836: 833: 803: 784: 760: 747: 744:CMCT methods 710: 693: 675: 662: 653: 644: 631: 617: 598: 590: 562: 553:ring opening 550: 542: 526: 507: 495: 484: 476: 440: 432: 425: 407: 379: 361: 352: 343: 330: 317: 314:m6A-LAIC-seq 284:chimeric DNA 281: 277: 265: 261:m6A-LAIC-seq 257: 231: 227: 209:proteinase K 201:growth media 191: 177: 173: 169: 165: 161: 150: 102: 58: 17: 15: 2919:Nucleosides 2701:ChemBioChem 1853:1885/220056 1791:(11): 215. 1785:Genome Biol 957:epigenomics 892:Nuclease P1 857:Apart from 845:Limitations 757:Limitations 672:Limitations 650:Limitations 628:Limitations 578:Myc epitope 529:overexpress 171:positives. 131:, and long 2908:Categories 2844:2019-12-09 2303:Chem. Biol 2213:IUBMB Life 1902:Genome Res 1685:(2): e12. 1004:References 988:(m6A) and 912:, such as 841:S2 cells. 830:hMeRIP-seq 820:methionine 720:eukaryotes 493:platform. 443:Drosophila 349:Nm-REP-seq 307:RNase T1/A 303:DNA ligase 188:PA-m6A-seq 2393:Mol. Cell 2262:Mol. Cell 2176:1088-9051 1653:253266867 1082:0021-9525 996:(HMM) or 728:ribosomal 491:Roche 454 325:spike ins 109:thymidine 105:adenosine 2887:29334379 2835:Archived 2831:23113984 2775:26816380 2731:25676849 2682:20224293 2660:RNA Biol 2605:25192136 2521:21976061 2472:19820108 2423:22099312 2374:26285676 2325:17113994 2284:20227365 2243:20561376 2235:10902565 2194:31427386 2129:25855956 2056:20835228 1983:15534692 1932:24407955 1862:26541084 1817:24286375 1766:27313037 1709:19059995 1645:36323972 1609:34893873 1559:27376769 1510:24141618 1461:26645578 1417:26121403 1349:26404942 1281:24269006 1204:22575960 1156:22608085 1100:27044895 812:isotopes 799:kingdoms 787:oxidised 732:splicing 563:miCLIP ( 451:cytosine 235:peptides 2895:3589823 2822:3491402 2753:Science 2722:4471624 2641:8373778 2596:4224642 2575:Bibcode 2512:4722041 2463:2794176 2414:3222873 2365:5694666 2185:6724681 2137:5325404 2094:9264612 2018:9264612 1923:3941116 1808:4053770 1757:5094196 1736:Bibcode 1728:Science 1700:2632927 1600:8728126 1550:5704921 1501:3884656 1408:4487409 1340:4604345 1272:3956118 1212:3517716 1184:Bibcode 1147:3383396 1091:4828693 910:lncRNAs 822:--> 804:in vivo 791:mammals 600:Inosine 537:5-aza-C 533:epitope 508:Aza-IP 455:uridine 447:DNA m5C 428:m5C-RIP 323:(ERCC) 295:RNase H 274:SCARLET 248:SCARLET 241:during 81:inosine 2893: 2885: 2829: 2819: 2773: 2729: 2719: 2680: 2639: 2603: 2593: 2567:Nature 2519: 2509: 2470: 2460: 2421: 2411: 2372: 2362: 2323: 2282: 2241: 2233: 2192: 2182: 2174: 2135: 2127: 2092: 2054: 2016: 1981: 1974:526178 1971: 1930: 1920: 1860: 1815: 1805: 1764: 1754: 1707: 1697: 1651: 1643: 1607: 1597: 1557: 1547: 1508: 1498: 1459: 1415: 1405: 1347: 1337: 1279: 1269: 1210: 1202: 1176:Nature 1154: 1144: 1098: 1088: 1080: 916:and Ψ 882:2D-TLC 767:primer 559:miCLIP 504:Aza-IP 457:, but 390:miCLIP 371:5’ cap 367:polyA+ 113:uracil 83:, and 2891:S2CID 2863:(PDF) 2838:(PDF) 2793:(PDF) 2239:S2CID 2133:S2CID 1649:S2CID 1208:S2CID 906:mRNAs 816:MS/MS 621:rtPCR 584:with 569:NSUN2 545:lysed 522:DNMT2 518:NSUN2 453:to a 291:ssDNA 133:ncRNA 129:snRNA 125:polyA 115:, so 46:rRNAs 42:tRNAs 2883:PMID 2827:PMID 2771:PMID 2727:PMID 2678:PMID 2637:PMID 2601:PMID 2517:PMID 2468:PMID 2419:PMID 2370:PMID 2321:PMID 2280:PMID 2231:PMID 2190:PMID 2172:ISSN 2125:PMID 2090:PMID 2052:PMID 2014:PMID 1979:PMID 1928:PMID 1858:PMID 1836:1859 1813:PMID 1762:PMID 1705:PMID 1641:PMID 1605:PMID 1555:PMID 1506:PMID 1457:PMID 1413:PMID 1345:PMID 1277:PMID 1251:Cell 1200:PMID 1152:PMID 1126:Cell 1096:PMID 1078:ISSN 908:and 861:and 808:mice 520:and 44:and 30:mRNA 2914:RNA 2875:doi 2817:PMC 2807:doi 2761:doi 2757:351 2717:PMC 2709:doi 2668:doi 2629:doi 2591:PMC 2583:doi 2571:515 2544:doi 2507:PMC 2499:doi 2458:PMC 2450:doi 2409:PMC 2401:doi 2360:PMC 2352:doi 2311:doi 2270:doi 2221:doi 2180:PMC 2164:doi 2117:doi 2082:doi 2044:doi 2006:doi 1969:PMC 1959:doi 1918:PMC 1910:doi 1848:hdl 1840:doi 1803:PMC 1793:doi 1752:PMC 1744:doi 1732:352 1695:PMC 1687:doi 1633:doi 1595:PMC 1587:doi 1545:PMC 1537:doi 1496:PMC 1488:doi 1480:RNA 1447:doi 1443:283 1403:PMC 1395:doi 1335:PMC 1327:doi 1267:PMC 1259:doi 1255:155 1192:doi 1180:485 1142:PMC 1134:doi 1130:149 1086:PMC 1070:doi 1066:213 914:m6A 795:TET 531:an 384:at 111:or 16:In 2910:: 2889:. 2881:. 2871:15 2869:. 2865:. 2833:. 2825:. 2815:. 2803:13 2801:. 2795:. 2769:. 2755:. 2751:. 2739:^ 2725:. 2715:. 2705:16 2703:. 2699:. 2676:. 2662:. 2658:. 2635:. 2625:32 2623:. 2599:. 2589:. 2581:. 2569:. 2565:. 2542:. 2538:. 2515:. 2505:. 2493:. 2489:. 2466:. 2456:. 2446:37 2444:. 2440:. 2417:. 2407:. 2397:44 2395:. 2391:. 2368:. 2358:. 2348:16 2346:. 2342:. 2319:. 2307:13 2305:. 2301:. 2278:. 2266:37 2264:. 2260:. 2237:. 2229:. 2217:49 2215:. 2211:. 2188:. 2178:. 2170:. 2160:29 2158:. 2154:. 2131:. 2123:. 2113:10 2111:. 2088:. 2078:36 2076:. 2064:^ 2050:. 2038:. 2026:^ 2012:. 2002:36 2000:. 1977:. 1967:. 1953:. 1949:. 1926:. 1916:. 1906:24 1904:. 1900:. 1888:^ 1870:^ 1856:. 1846:. 1834:. 1811:. 1801:. 1789:14 1787:. 1783:. 1760:. 1750:. 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Knowledge (XXG)

Epitranscriptomic sequencing

Index