EpiNext CUT&RUN RNA m6A-Seq Kit | P-9016

The EpiNext™ CUT&RUN RNA m6A-Seq Kit is a complete set of optimized buffers and reagents designed to enrich an RNA fragment containing m6A from low input RNA and to prepare a library for next generation sequencing (m6A-seq/meRIP-seq) using Illumina platforms such as Illumina Genome Analyzer II, HiSeq and MiSeq systems. 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 meRIP 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. This kit has the following advantages and features: 

• High enrichment: Use RNA cleavage enzyme mix to simultaneously fragment RNA and cleave/remove any RNA sequences at 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 the maximal protection of the target m6A-containing regions and the minimized sample loss, allowing the input RNA to be as low as 500 ng.
• Minimal Background: Cleavage of unbound RNA sequences in the two ends of the target m6A-containing sequences enables the minimized MeRIP/sequencing background, allowing data analysis with <10 million reads. 
• Fast, streamlined procedure: The procedure from RNA to library cDNA is less than 6 hours with <1 h of hands-on time. 
• Highly convenient: The kit contains all required components for each step of the CUT&RUN RNA m6A -Seq, which are sufficient for both m6A-containing RNA sequence capture and captured cDNA library preparation, thereby allowing CUT&RUN RNA m6A –Seq 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-Seq starting from total RNA. In the reaction, RNA sequences at 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 target m6A-containing RNA fragments are then reverse-transcripted ligated with adaptors. The ligated RNA is released, purified, and amplified with a high-fidelity PCR mix for library cDNA construction. The cDNA is then cleaned, released, and eluted. Included in the kit is a negative control non-immune IgG, which can demonstrate the efficacy of the kit and performance at the enriched RNA quantification or bioanalyzer analysis 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 EpiNext CUT&RUN RNA m6A-Seq Kit combines the advantages CUT&RUN with the fastest procedures in a convenient all-in-one 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 be 1 µg to 10 µg. For optimal preparation, the input amount should be 5 µg cells, although this CUT&RUN RNA m6A -Seq data can be obtained with the lowest amount of total RNA as less as 500 ng.