Total RNAs were extracted and purified by using PolyTtract mRNA Isolation System (Promega, Hong Kong). After fragmentation, RNA was incubated with m6A antibody for immunoprecipitation according to the standard protocol of the Magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany). Enrichment of m6A containing mRNA was then analyzed either through RT-qPCR or high-throughput sequencing. Primers to m6A negative region of EEF1A was used as the negative control and primers to m6A postive region of EEF1A was used as the positive control according to the standard protocol of the Magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany). For high-throughput sequencing, purified RNA fragments were used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (New England BioLabs) and were sequenced with Illumina HiSeq X Ten platform. Library preparation and high-throughput sequencing were performed by Novogene (Beijing, China).
Magna methylated rna immunoprecipitation m6a kit
Manufactured by Merck Group
Sourced in Germany, United States
The Magna methylated RNA immunoprecipitation m6A Kit is a tool designed for the enrichment and detection of N6-methyladenosine (m6A)-modified RNA. It enables the isolation and purification of m6A-containing RNA fragments from total RNA samples, facilitating the study of this important RNA modification.
Automatically generated - may contain errors
Lab products found in correlation
Polyttract mrna isolation system, by Promega (2 mentions)
Anti m6a antibody, by Merck Group (1 mentions)
Magna chip protein a g magnetic beads, by Merck Group (1 mentions)
M6a antibody, by New England Biolabs (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Rna purification kit, by Qiagen (1 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
5 protocols using magna methylated rna immunoprecipitation m6a kit
1
Profiling m6A-Enriched mRNA by RNA Immunoprecipitation
2
Magna MeRIP-qPCR Protocol for m6A Profiling
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The Magna Methylated RNA Immunoprecipitation m6A Kit (Millipore, Massachusetts, USA) was employed to investigate the m6A modification of specific genes based on the manufacturer’s guidelines. A total of 5 μg of anti-m6A antibody (CS220007, Millipore, Massachusetts, USA) or regular mouse IgG (CS200621, Millipore, Massachusetts, USA) was subjected to a pre-washing step and then treated at room temperature with Magna ChIP protein A/G magnetic beads (CS203152, Millipore, Massachusetts, USA) for a duration of 1 h. Next, the complexes formed by the antibodies and beads were mixed with pure poly-(A) RNA and subjected to RT-qPCR analysis to examine the enrichment of m6A-modified mRNA. The relative m6A enrichment in each specimen was determined by normalizing to the input. The sequences of the primer utilized for MeRIP-qPCR are provided in Table S1 .
3
Quantifying m6A-modified mRNA
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Cells were mixed with the RIP lysis buffer and immunoprecipitated by co-incubation with an m6A antibody using a Magna methylated RNA immunoprecipitation m6A Kit (Millipore, USA). The enrichment of mRNA containing m6A was then analyzed utilizing RT-qPCR.
4
Quantifying m6A-Containing mRNA
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Total RNAs were extracted from stable ALKBH5 knockdown and catalytic inactive mutation C918 cells and their corresponding control cells. Chemically fragmented RNA (approximately 100 nucleotides) was incubated with m6A antibody (New England BioLabs) for immunoprecipitation according to the protocol of the Magna methylated RNA immunoprecipitation m6A kit (Merck Millipore). Enrichment of m6A-containing mRNA was analyzed using qRT-PCR.
5
M6A RNA Enrichment and Sequencing
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Total RNA was extracted and purified by PolyTtract mRNA Isolation System (Promega). After fragmentation, magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany) was used for m6A RNA immunoprecipitation. In short, total RNA was dispersed into 100nt-long oligonucleotides and then incubated with anti-m6A antibodies or immunoglobulin (IgG). The fragmented RNA containing m6A was eluted and purified with RNA purification kit (Qiagen, Germany). Enriched fragments were analyzed by qPCR. The purified RNA fragments were used to construct libraries using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs, USA) and sequenced using Illumina HiSeq 2000 (Illumina, USA) platform. The level of m6A mRNA was measured by qPCR.
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