N6 methyladenosine

Manufactured by Merck Group

Sourced in United States

N6-methyladenosine is a modified nucleoside found in various types of RNA. It plays a role in the regulation of RNA metabolism and function. This product is intended for use in research applications.

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Lab products found in correlation

1 methyladenosine, by Merck Group (3 mentions) Dynabeads protein a, by Thermo Fisher Scientific (3 mentions) Uridine, by Merck Group (2 mentions) Cytidine, by Merck Group (2 mentions) 5 methylcytidine, by Merck Group (2 mentions) 5 methyluridine, by Merck Group (2 mentions) Hiseq x, by Illumina (2 mentions) Protein a beads, by Repligen (2 mentions) Anti m6a polyclonal antibody, by Synaptic Systems (2 mentions) Mrna sequencing kit, by Illumina (2 mentions) Nextseq 550, by Illumina (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Dynabeads oligo, by Thermo Fisher Scientific (2 mentions) Anti m6a rabbit polyclonal antibody, by Synaptic Systems (2 mentions) Rna clean and concentrator 5, by Zymo Research (2 mentions) Scriptseq v2 kit, by Illumina (2 mentions) M per buffer, by Thermo Fisher Scientific (2 mentions) Rabbit igg control bound to protein a dynabeads, by Thermo Fisher Scientific (2 mentions) Pseudouridine, by Merck Group (2 mentions) Xanthosine, by Merck Group (2 mentions)

Close spelling variants of this product

N6-methyladenosine 5-monophosphate sodium salt (10 citations) Anti-N6-methyladenosine human monoclonal antibody (2 citations)

17 protocols using n6 methyladenosine

Nucleoside Analysis Protocol

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Guanosine
(Guo), cytidine (Cyt), adenosine (Ado), uridine (Urd), 5-methylcytidine
(5-me-Cyt), 5-methyluridine (5-me-Urd), N⁶-methyladenosine (N⁶-me-Ado), 2′-deoxyguanosine (dG), 2′-deoxycytidine
(dC), 2′-deoxyadenosine (dA), thymidine (dT), 5-methyl-2′-deoxycytidine
(5-me-dC), N⁶-methyl-2′-deoxyadenosine
(N⁶-me-dA), 8-oxo-7,8-dihydro-2′-deoxyguanosine
(8-oxo-dG), nucleosides test mix [containing cytidine (Cyt), guanosine
(Guo), adenosine (Ado), uridine (Urd), inosine (Ino), 5-methylcytidine
(5-me-Cyt), 2′-O-methylcytidine (2-O-me-Cyt), 3-methylcytidine methosulfate (3-me-Cyt), 7-methylguanosine
(7-me-Guo), 1-methyladenosine (1-me-Ado), 5-methyluridine (5-me-Urd),
β-pseudouridine (β-Urd), 2-thiocytidine dihhydrate (ThioC)],
DNA from calf thymus (ctDNA) sodium salt, nuclease P₁ from Penicillium citrinum (NP1), phosphodiesterase I from Crotalus adamanteus (snake) venom (SVPDE), alkaline
phosphatase from Escherichia coli (AKP),
ammonium acetate, ammonium bicarbonate, tris(hydroxymethyl)aminomethane
(Tris-buffer, pH 7.4), zinc chloride, and formic acid were obtained
from Sigma-Aldrich (St. Louis, MO). Chelex-100 resin was purchased
from Bio-Rad (Solna, Sweden). All the solvents used were of HPLC grade.
Experiments containing nucleic acids were carried out in DNA LoBind
tubes, 1.5 mL (Eppendorf).

Martella G., Motwani N.H., Khan Z., Sousa P.F., Gorokhova E, & Motwani H.V. (2023). Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis. Chemical Research in Toxicology, 36(9), 1471-1482.

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m6A RNA Immunoprecipitation and Sequencing

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Polyadenylated RNA, separated from total RNA using Oligo dT Magnetic Beads (Vazyme, N403), was fragmented in 1X NEB Next Magnesium RNA Fragmentation Buffer at 94 °C for 5 min. 10 ng of fragmented RNA was saved as input. 10 μg fragmented RNA was further incubated with 5 μg rabbit anti-m⁶A polyclonal antibody (Synaptic Systems, catalog number 202003) in IPP buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.1% Igepal CA-630) overnight at 4 °C. The m⁶A-Ab mixture was then immunoprecipitated by incubation with protein-G magnetic beads at 4 °C for another 2 hours. RNA was then eluted from the beads with IPP buffer containing 0.5 mg/ml N6-methyladenosine (Sigma-Aldrich, M2780). VAHTS stranded mRNA-seq library prep kit (Vazyme, NR601) was used for library construction. Libraries were sequenced on HiSeq X (Illumina) to produce paired-end 150 bp reads (Supplementary Data 6).

Liu J., Huang T., Yao J., Zhao T., Zhang Y, & Zhang R. (2023). Epitranscriptomic subtyping, visualization, and denoising by global motif visualization. Nature Communications, 14, 5944.

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m6A Methylation Profiling by meRIP-Seq

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meRIP-Seq and data analysis were performed as previously described with minor modifications (Dominissini et al., 2013 (link)). Briefly, mRNA was first purified from total RNA using PolyATtract® mRNA Isolation Systems (Promega). Then 5 μg mRNA was fragmented and immunoprecipated with anti-m⁶A antibody (Synaptic Systems, 202003), the immunoprecipated RNA was washed and eluted by competition with N6-Methyladenosine (Sigma-Aldrich, M2780). The purified RNA fragments from m⁶A meRIP were used for library construction using TruSeq Stranded mRNA Sample Prep Kits (Illumina RS-122-2101) and sequenced with Illumina HiSeq 2000. Reads mapping, m⁶A peak calling and motif search were performed as described (Dominissini et al., 2013 (link)). Analysis of the published PAR-CLIP was performed as previously described (Liu et al., 2014 (link)). For deep sequence analysis of RNA expression, 2 μg total RNA was used for the library construction using TruSeq Stranded mRNA Sample Prep Kits (Illumina RS-122-2101) according to the manufacturer’s instructions.

Lin S., Choe J., Du P., Triboulet R, & Gregory R.I. (2016). METTL3 promotes translation in human cancer cells. Molecular cell, 62(3), 335-345.

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m6A-seq of chemically fragmented RNA

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Total RNA (>300 µg) of samples were chemically fragmented into ~100-nucleotide-long fragments by 5 min incubation at 94 °C in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7). The fragmentation reaction was stopped with 0.05 M EDTA, followed by standard ethanol precipitation. Samples were resuspended in H₂O at ~1 mg ml/1 concentration and subjected to m⁶A-seq. Fragmented RNA was incubated for 2 h at 4 °C with 5 mg of affinity-purified anti-m⁶A polyclonal antibody (Synaptic Systems cat. no. 202 003) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4). The mixture was then immunoprecipitated with protein-A beads (Repligen) at 4 °C for an additional 2 h. After extensive washing, bound RNA was eluted from the beads with 0.5 mg ml/1 N⁶-methyladenosine (Sigma-Aldrich) in IPP buffer, and ethanol precipitated. RNA was resuspended in H₂O and used for library generation with the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was carried out on Illumina HiSeq2500 according to the manufacturer’s instructions, using 10 pM template per sample for cluster generation, and sequencing kit V2 (Illumina). These raw data are available at NCBI SRA series PRJNA701370 (public release date set to 2021-08-01).

Ben-Haim M.S., Pinto Y., Moshitch-Moshkovitz S., Hershkovitz V., Kol N., Diamant-Levi T., Beeri M.S., Amariglio N., Cohen H.Y, & Rechavi G. (2021). Dynamic regulation of N6,2′-O-dimethyladenosine (m6Am) in obesity. Nature Communications, 12, 7185.

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m6A-Seq Protocol for RNA Methylation

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Fragmented RNA (2.5 mg total RNA) was incubated for 2 h at 4 °C with 5 μg of affinity purified anti-m⁶A polyclonal antibody (Synaptic Systems, Göttingen, Germany) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4). The mixture was then immunoprecipitated by incubation with protein-A beads (Repligen, Waltham, MA, USA) at 4 °C for an additional 2 h. After extensive washing, bound RNA was eluted from the beads with 0.5 mg/ml N⁶-methyladenosine (Sigma-Aldrich, St Louis, MO, USA) in IPP buffer and ethanol precipitated. The RNA was resuspended in H₂O and used for library generation with mRNA sequencing kit (Illumina Inc., San Diego, CA, USA).

Tao X., Chen J., Jiang Y., Wei Y., Chen Y., Xu H., Zhu L., Tang G., Li M., Jiang A., Shuai S., Bai L., Liu H., Ma J., Jin L., Wen A., Wang Q., Zhu G., Xie M., Wu J., He T., Huang C., Gao X, & Li X. (2017). Transcriptome-wide N6-methyladenosine methylome profiling of porcine muscle and adipose tissues reveals a potential mechanism for transcriptional regulation and differential methylation pattern. BMC Genomics, 18, 336.

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m6A-SMART-seq for Mouse Dentate Gyrus Transcriptome

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Total RNA from adult mouse DG was isolated using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen). mRNA purification was performed with poly(A)+ RNA selection twice using Dynabeads Oligo (dT)25 (Thermo Fisher; 61006). A total of 150 ng of mRNA was subjected to m⁶A-SMART-seq using anti-m⁶A rabbit polyclonal antibody (Synaptic Systems, 202003) as previously described^{9 (link)}. Briefly, 5 μg of anti-m⁶A polyclonal antibody was conjugated to Dynabeads Protein A (Thermo Fisher; 10001D) and used for each affinity pull-down. The m⁶A RNA was eluted twice with 6.7 mM N⁶-Methyladenosine (Sigma-Aldrich; M2780) in 1 × IP buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% (vol/vol) Igepal CA-630) and recovered by RNA Clean and Concentrator-5 (Zymo Research). Libraries were generated using the SMART-seq protocol as described^{35 (link)}. Three biological replicates for each condition were sequenced using Illumina NextSeq 550 from a single end for 75 bases.

Shi H., Zhang X., Weng Y.L., Lu Z., Liu Y., Lu Z., Li J., Hao P., Zhang Y., Zhang F., Wu Y., Delgado J.Y., Su Y., Patel M.J., Cao X., Shen B., Huang X., Ming G.L., Zhuang X., Song H., He C, & Zhou T. (2018). m6A facilitates hippocampus-dependent learning and memory through Ythdf1. Nature, 563(7730), 249-253.

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m6A-RNA Immunoprecipitation and Sequencing

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Biological replicates of control and TARBP2 knockdown cells (MDA-LM2 background) were collected and processed as previously described (Alarcón et al., 2015b (link)). 1×10⁷ cells per sample were lysed using LB1 buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Triton X-100) and 1X protease inhibitor cocktail (Thermo Scientific). The nuclear fraction was then lysed with M-PER buffer (Thermo Scientific) and diluted tenfold in dilution buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl) before the immunoprecipitation. Rabbit anti-m6A antibody (Synaptic Systems) and rabbit IgG control bound to protein A dynabeads (Invitrogen) were used for the immunoprecipitations. The immunoprecipitated RNA was eluted with N6-methyladenosine (Sigma-Aldrich), ethanol precipitated and resuspended in water. RNA was barcoded using ScriptSeq V2 kit (Epicentre) and sequenced at Rockefeller University Genomics Core.

Fish L., Navickas A., Culbertson B., Xu Y., Nguyen H.C., Zhang S., Hochman M., Okimoto R., Dill B.D., Molina H., Najafabadi H.S., Alarcón C., Ruggero D, & Goodarzi H. (2019). Nuclear TARBP2 drives oncogenic dysregulation of RNA splicing and decay. Molecular cell, 75(5), 967-981.e9.

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m6A-RIP-Seq Profiling Protocol

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3×10⁷ cells/sample were lysed using LB1 buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton x-100, and protease inhibitors). The nuclear fraction was then lysed with M-PER buffer (Thermo Scientific) and diluted 10-fold in dilution buffer (50mM Tris-Cl, pH 7.4, 100mM NaCl) before the immunoprecipitation. Rabbit α-m6A antibody (Synaptic Systems) and rabbit IgG control bound to protein A Dynabeads (Invitrogen) were used for the immunoprecipitations. The immunoprecipitated RNA was eluted with N6-methyladenosine (Sigma-Aldrich), ethanol precipitated and resuspended in water. RNA was barcoded using ScriptSeq V2 kit (Epicentre) and sent for sequencing.

Alarcón C.R., Lee H., Goodarzi H., Halberg N, & Tavazoie S.F. (2015). N6-methyl-adenosine (m6A) marks primary microRNAs for processing. Nature, 519(7544), 482-485.

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m6A-SMART-seq for Mouse Dentate Gyrus Transcriptome

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m6A Immunoprecipitation and RNA Sequencing

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Peritoneal macrophages were isolated from ALKBH5 knockout or control littermate mice, and total RNA was isolated with RNA Purification Kit (EZBioscience, B0004D, China) as above described. RNA samples were chemically fragmented into,100-nucleotide-long fragments by 5 min incubation at 94 °C in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7). The fragmentation reaction was stopped with 0.05 M EDTA, followed by standard ethanol precipitation. Then the fragmented RNA (400 mg mRNA or 2.5 mg total RNA) was incubated for 2 hours at 4 °C with 5 mg of anti-m6A antibody (Abcam; Cat# ab151230) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM TrisHCl, pH 7.4) supplemented with RNase inhibitors. The mixture was then immunoprecipitated by Protein A/G beads (Repligen) at 4 °C for an additional 2 hours. After extensive washing, bound RNA was eluted from the beads with 0.5 mg/ml N6-methyladenosine (Sigma-Aldrich) in IPP buffer, and ethanol precipitated. RNA was resuspended in H₂O and used for library generation with mRNA sequencing kit (Illumina). m6A enrichment was determined by qPCR analysis with primers in the Supplemental Table 1.

Zhuang T., Chen M.H., Wu R.X., Wang J., Hu X.D., Meng T., Wu A.H., Li Y., Yang Y.F., Lei Y., Hu D.H., Li Y.X., Zhang L., Sun A.J., Lu W., Zhang G.N., Zuo J.L, & Ruan C.C. (2024). ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice. Nature Communications, 15, 1995.

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