EpiQuik CUT&RUN m6A RNA Enrich

The EpiQuik™ CUT&RUN m6A RNA Enrichment (MeRIP) Kit is a complete set of optimized buffers and reagents designed to enrich an RNA fragment containing m6A from low input RNA and to identify region-specific m6A by PCR or profile epitranscriptome-wide m6A by next-generation sequencing using Illumina platforms or other methods. The innovative working principle, optimized protocol, and components of the kit allow for the capture of the m6A fragment with minimal non-specific background levels. The enriched RNA is specifically suitable to construct both non-barcoded (singleplexed) and barcoded (multiplexed) libraries quickly, allowing m6A regions to be mapped with less bias and at a high resolution. The kit has the following features: 

• High enrichment: Uses an RNA cleavage enzyme mix to simultaneously fragment RNA and cleave/remove any RNA sequences in both ends of the target m6A-containing sequences without affecting RNA regions occupied by the antibody. Short RNA fragments are generated only bound with anti- m6A antibody. True target m6A-enriched regions can, therefore, be reliably identified, and high-resolution mapping achieved.
• Low input: Unbound RNA cleavage and immunocapture are processed in the same single-tube, which enables maximum protection of the target m6A-containing regions and minimized sample loss, allowing the input RNA to be as low as 500 ng.
• Minimal Background: Cleavage of unbound RNA sequences in the two (2) ends of the target m6A-containing sequences enables minimized MeRIP/sequencing background, allowing data analysis with <10 million reads.
• Fast, streamlined procedure: The procedure from RNA to enriched m6A RNA is around 2 hours and 30 min with <30 min of hands-on time
• Highly convenient: The kit contains all required components for each step of the EpiQuik™ CUT&RUN m6A RNA Enrichment Kit, which are sufficient for m6A-containing RNA sequence capture, thereby allowing CUT&RUN m6A RNA enrichment to be the most convenient with reliable and consistent results. 

Background Information
N6-methyl-adenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes. The m6A modification is catalyzed by a methyltransferase complex METTL3 and removed by m6A RNA demethylases FTO and ALKBH5, which catalyze m6A demethylation in an α-ketoglutarate (α-KG)- and Fe2+-dependent manner. METTL3, FTO, and ALKBH5 are known to play important roles in many biological processes, ranging from development and metabolism to fertility. m6A accounts for more than 80% of all RNA base methylations and exists in various species. m6A is mainly distributed in mRNA and also occurs in non-coding RNA, such as tRNA, rRNA, and snRNA. The relative abundance of m6A in mRNA transcripts has been shown to affect RNA metabolism processes such as splicing, nuclear export, translation ability and stability, and RNA transcription. Abnormal m6A methylation levels induced by defects in m6A RNA methylase and demethylase could lead to dysfunction of RNA and cause disease. For example, abnormally low levels of m6A in target mRNAs due to increased FTO activity in patients with FTO mutations, through an as-yet-undefined pathway, contributes to the onset of obesity and related diseases. The dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significance. Therefore, more useful information for a better understanding of m6A RNA methylation levels and distribution on RNA transcripts could benefit diagnostics and therapeutics of disease.

Principle & Procedure
This kit contains all necessary reagents required for carrying out a successful m6A RNA enrichment starting from total RNA. In the reaction, RNA sequences in both ends of the target m6A-containing regions are cleaved/removed and the target m6A-containing fragments are pulled down using a beads-bound m6A capture antibody. The enriched RNA is then released, purified and eluted. Included in the kit are a non-immune IgG control and m6A positive control. These can be used to demonstrate the efficacy of the kit and performance at the enriched RNA quantification or bioanalyzer step.

Cleavage under targets and release using nuclease (CUT&RUN) was recently developed as an advancement over standard ChIP methods, requiring lower amounts of input material and providing significantly improved mapping resolution. The EpiQuik CUT&RUN m6A RNA Enrichment (MeRIP) Kit combines the advantages of CUT&RUN with the fastest procedures in a convenient kit to reliably enrich RNA fragments containing the m6A modification from low input RNA, with minimal non-specific background levels. 

Starting Materials
The input material required is RNA, which can be isolated from any species and from various tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, paraffin-embedded tissues, blood, body fluid samples, etc. The amount of total RNA for each reaction can range from 1 µg to 20 µg. For optimal preparation, the input amount should be 10 µg RNA, although data can be obtained from as low as 500 ng of total RNA using this kit.