m6Am-Seq

The m6A modification is one of the common modifications in eukaryotic mRNA; m6Am, also an important RNA modification, is very similar to m6A in molecular structure. It was not until the gradual development of m6Am sequencing technology in recent years that paved the way for further in-depth study of m6Am. an important RNA modification.

Overview

On the basis of m6A modification, the 2'-hydroxyl of ribose of the same adenylate residue is also methylated to generate a 2'-methoxy structure (2'-O-CH₃) of m6Am. Unlike the common distribution of m6A, the distribution of m6Am modifications is largely restricted – presenting at the first transcribed nucleotide in ∼30% of all cellular mRNAs, m6Am can affect the fate of a large subset of the transcriptome. And the locus of m6Am modification is highly conserved, whether found in zebrafish, mouse, or human cells. The m6Am modification is a reversible dynamic modification. When cells encounter stress stimuli such as heat shock and hypoxia, the m6Am level increases, indicating that m6Am may play an important role in the cellular stress response. Recent studies have also found that in addition to the preceding coding nucleotide residues of mRNA, there is also a distribution of m6Am methylation modifications within lncRNA and snRNA.

CD Genomics' m6Am-seq sequencing service uses 5' exonuclease to eliminate the signal interference of m6A modification on unintended RNA fragments, and selectively obtains the sequence information of m6Am modification-enriched regions downstream of the 5'-cap structure of mRNAs and non-coding RNAs. Next, we performed reverse transcription library construction and high-throughput sequencing of the selectively obtained m6Am-modified RNA fragments (sequencing results will include both mRNA and non-coding RNA). CD Genomics can help you generate transcriptome-wide maps that distinguish m6Am and m6A. Through subsequent bioinformatics analysis, the sequenced fragments from mRNAs and non-coding RNAs can be distinguished, thereby comprehensively revealing the m6Am modification sites of various RNAs, and predicting their possible biological functions.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Statistics of m6Am distribution
• Peak calling and annotation at whole-transcriptome level
• Identify m6Am in mRNA, lncRNA, and snRNA
• Clustering analysis and enrichment analysis
• Transcriptome-wide profiling of m6Am methylation
• Differential analysis
• Motif identification of enrichment sites
• Evolutionary conservation analysis
• GO and Pathway analysis
• Explore new m6Am methylation sites

Sample Requirements

Tissue sample: 100 mg – 1 g

Cell sample: cell amount: above 2×10⁷

RNA sample: RNA quantity: 30-300 μg; RNA purity: OD_260/280 = 1.6~2.3; OD_260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5 or RIN ≥ 7

Please make sure that the RNA is not significantly degraded.

Sample storage: Cell samples or fresh tissue pieces (cut into 5-10 mg pieces) can be treated with TRIzol or RNA protectant, frozen in liquid nitrogen, and stored at -80°C. RNA samples can be dissolved in ethanol or RNA-free ultrapure water, and stored at -80°C. Avoid repeated freezing and thawing during sample storage.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

1. Jiang J, Song B, Chen K, et al. m6AmPred: Identifying RNA N6, 2'-O-dimethyladenosine (m6Am) sites based on sequence-derived information. Methods. 2021 Feb 2.
2. Boulias K, Toczydłowska-Socha D, Hawley BR, et al. Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome. Molecular cell. 2019 Aug 8;75(3).