Total RNA was extracted from cells using TRIzol™ LS Reagent (Thermo Fisher Scientific #10296010) or RNeasy Plus Mini Kit (Qiagen #74134). mRNA was isolated and purified using the Dynabeads™ mRNA purification kit (for mRNA purification from total RNA preperations; Invitrogen #61006) following the manufacturer's instructions. For the m6A dot blot, mRNA was hybridized onto the Hybond-N+ membrane (GE Healthcare). After crosslinking spotted mRNA to the membrane using a Stratalinker 2400 UV Crosslinker, the membrane was blocked with 5% skimmed milk for 1 h, and incubated with mouse anti-m6A antibody (1:1000, Millipore #MABE1006) at 4°C overnight. Then the membrane was incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse-IgG at room temperature for 1 h. The membrane was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% Methylene Blue. The relative m6A level was quantified using ImageJ.
Mabe1006
Manufactured by Merck Group
Sourced in United States, Germany
MABE1006 is a laboratory equipment designed for general scientific applications. It is a versatile and reliable instrument that can be used for various tasks in research and testing environments. The core function of MABE1006 is to provide precise and accurate measurements, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Magna merip m6a kit, by Merck Group (4 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Ecl imaging system, by Bio-Rad (2 mentions)
Hybond, by Cytiva (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
N6 methyladenosine 5 monophosphate sodium salt, by Chem-Impex (2 mentions)
Actinomycin d, by Bio-Techne (1 mentions)
Ab205606, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico plus kit, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti mettl3, by Fortis Life Sciences (1 mentions)
Anti brd4 n term, by Abcam (1 mentions)
Anti brd4 c term, by Fortis Life Sciences (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
9 protocols using mabe1006
1
Quantifying m6A Methylation in mRNA
2
m6A Methylation Profiling Protocol
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Cells with stable knockdown of WTAP were subjected to MeRIP assay using Magna MeRIP™ m6A Kit (17–10,499, Millipore, MA) in accordance with manufacturer’s recommendations. In short, 200 μg total RNA was isolated and randomly fragmented into 100 nucleotides or less, followed by the immunoprecipitation with 10μg m6A antibody (MABE1006, Millipore) or anti-mouse IgG which was linked to Magna ChIP Protein A/G Magnetic Beads. To determine the appropriate ratio between RNA and antibody, a dilution assay had been applied to optimize the MeRIP system and ensure the m6A antibody was not saturated. And one-tenth volume of fragmented RNA was saved as “10% input”. Elution of m6A-precipitated RNA was based on 6.7 mM N6-methyladenosine 5′-monophosphate sodium salt. And modification of m6A towards particular genes was determined by qPCR analysis with specific primers (All primers for MeRIP-qPCR were listed in Additional file 1 : Table S1) Note: m6A sites of specific genes were predicted in RMBase v2.0 (http://rna.sysu.edu.cn/rmbase/ ) and SRAMP (http://www.cuilab.cn/sramp ) (Additional file 5 : Data S1). We focused on the potential m6A sites in 3′ UTR near the stop codon and designed primers to ensure that target sequence embodied all these sites with the limited length of 100 nt.
3
Methylation-specific RNA Immunoprecipitation (MeRIP)
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The MeRIP assay was performed according to the manufacturer's instructions of Magna MeRIP™ m6A Kit (17‐10499; Millipore, Billerica, Massachusetts, USA). In brief, RNA was chemically fragmented into 100 nucleotides or smaller fragments followed by magnetic immunoprecipitation with a monoclonal antibody (1:1000, MABE1006, Millipore) toward m6A. After washing with IP buffer, the RNA was eluted and precipitated with ethanol. Then, the isolated RNA fragments could be subjected with qRT‐PCR. The antibodies used in this study are listed in Supplementary Table S1 .
4
m6A RNA Methylation Analysis
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Cells were treated with 5µg/ml of actinomycin D (Tocris #1229). At indicted time point 3, 6, and 9 hours total RNA was extracted and DMSO treated cells was used as a control, and relative RNA expression was detected by qPCR m6A dot blot Total RNA was extracted from cells using TRIzol™ LS Reagent (Thermo scientific 10296010) or RNeasy Plus Mini Kit (Qiagen# 74134). mRNA was isolated and purified using Dynabeads™ mRNA Purification Kit (for mRNA purification from total RNA preps) (Invitrogen # 61006) following the manufacturer's instructions. For m6A dot blot, mRNA was hybridized onto the Hybond-N+ membrane (GE Healthcare). After crosslinking spotted mRNA to membrane using Stratalinker 2400 UV Crosslinker, the membrane was blocked with 5% skimmed milk for 1 h, incubated with mouse anti-m6A antibody (1:1000, Millipore # MABE1006) at 4°C overnight. Then the membrane was incubated with HRP-conjugated donkey anti-mouse IgG at room temperature for 1 h. The membrane was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% methylene blue. Relative m6A level was quantified using ImageJ.
5
m6A-Specific Methylation Enrichment
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Magna MeRIP m6A Kit (Millipore, USA) was used to conduct the MeRIP assay in accordance with the manufacturer’s instructions [18 (link)]. Anti-m6A body (MABE1006, Merck Millipore), anti-METTL3 body (ab195352, Abcam, USA) and anti-DDDK tag body ((ab205606, Abcam, USA) were applied for MeRIP detection. After washing with IP buffer, we eluted and subject RNA to the ethanol precipitation. Then, qRT-PCR was conducted to measure the enrichment of m6A containing mRNA.
6
m6A RNA Immunoprecipitation Protocol
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m6A Ribonucleoprotein Immunoprecipitation reactions were performed by first isolating PolyA+ RNA from normoxic and hypoxic cells. Protein G Dynabeads (Thermo Fisher Scientific, Baltics UAB) were washed 3× in 1 mL of IPP buffer (10 mM Tris-HCL pH7.4, 150 mM NaCl, 0.1% NP-40). 25 μl of beads required per IP. Anti-N6-methyladenosine mouse monoclonal antibody (EMD Millipore, Temecula, CA, MABE1006) was added to the beads (5 μg/IP) and brought up to 1mL with IPP buffer. Bead mixture was tumbled for 16 hours at 4° C. Beads were washed 5× with IPP buffer and 100 ng of PolyA+ RNA was added to the beads along with 1 mM DTT and RNase out. The mixture was brought up to 500 μl with IPP buffer. Bead mixture was tumbled at 4° C for 4 hours. Beads were washed 2× in IPP buffer, placed in to a fresh tube, and washed 3× more in IPP buffer. m6A RNA was eluted off the beads by tumbling 2× with 125 μl of 2.5 mg/mL N6-Methyladenosine-5′-monophosphate sodium salt (CHEM-IMPEX INT'L INC., Wood Dale, IL). Supernatant was added to Trizol-LS followed by RNA isolation as per manufacture's protocol. Final RNA sample was brought up in 10 μl of water.
7
m⁶A RNA Immunoprecipitation and Elution
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m6A ribonucleoprotein immunoprecipitation reactions were performed by first isolating poly(A)+ RNA from normoxic and hypoxic cells. Protein G Dynabeads (Thermo Fisher Scientific) were washed 3× in 1 mL of IPP buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl, 0.1% NP-40). Twenty-five microliters of beads were required per IP. Anti-N6-methyladenosine mouse monoclonal antibody (EMD Millipore, MABE1006) was added to the beads (5 µg/IP) and brought up to 1 mL with IPP buffer. As a negative control, beads without antibody were used as well. Bead mixture was tumbled for 16 h at 4°C. Beads were washed 5× with IPP buffer and 100 ng of poly(A)+ RNA was added to the beads along with 1 mM DTT and RNase out. The mixture was brought up to 500 µL with IPP buffer. Bead mixture was tumbled at 4°C for 4 h. Beads were washed 2× in IPP buffer, placed into a fresh tube, and washed 3× more in IPP buffer. m6A RNA was eluted off the beads by tumbling 2× with 125 µL of 25 mg/mL N6-methyladenosine-5′-monophosphate sodium salt (Chem-Impex International Inc.). Supernatant was added to TRIzol-LS followed by RNA isolation as per manufacturer's protocol. The final RNA sample was brought up in 10 µL of water.
8
Mapping m6A Epitranscriptomic Landscape
9
m6A Immunoprecipitation and Quantification
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The purified mRNA fragments were incubated with the m6A antibody for immunoprecipitation using the Magna MeRIP™ m6A kit (#17–10,499, Merck Millipore, Germany). Briefly, 30 μg of the total RNA was collected and mixed with the MeRIP reagent. The previously resuspended Magna ChIP protein A/G Magnetic Beads were conjugated with anti-m6A (MABE1006, Merck Millipore, Germany) or normal mouse anti-IgG antibodies (CS200621, A&D Technology, Japan) overnight at 4°C. Finally, the magnetic beads were eluted from the complex, and the RNA level was quantified using RT-qPCR.
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