m6Am-Exo-seq Service

m6Am-Exo-seq is a specialized sequencing technique that employs exonuclease digestion followed by immunoprecipitation to selectively map and quantify the cap-proximal m6Am RNA modification, distinguishing it from internal m6A sites. CD BioSciences offers expert, end-to-end m6Am-Exo-seq services, delivering precise insights into this pivotal epitranscriptomic mark for researchers investigating mRNA stability, translation initiation, and cellular signaling.

Overview of m6Am-Exo-seq

m6Am-Exo-seq is an advanced next-generation sequencing technique specifically engineered for the selective identification and quantification of m6Am, a modification occurring at the first transcribed nucleotide adjacent to the mRNA cap. Its core methodology involves the treatment of RNA with a 5'→3' exonuclease (such as XRN-1) to degrade RNA species lacking a protective cap structure, thereby enriching for intact, capped transcripts. The enriched pool is then subjected to immunoprecipitation using an antibody specific to m6A/m6Am, and the captured RNA is sequenced. This two-step enrichment strategy of exonuclease digestion followed by immunoprecipitation ensures that the sequenced signals predominantly originate from genuine cap-associated m6Am modifications rather than internal m6A. This technique is critical for elucidating the unique role of m6Am in regulating mRNA cap homeostasis, translation efficiency, and transcript stability.

m6Am-seq combines RNA immunoprecipitation with optimized demethylation reactions to enrich and distinguish m6Am and 5'-UTR m6A.

Fig.1 m6Am-seq combines RNA immunoprecipitation with an optimized demethylation reaction to enrich and distinguish m6Am and 5'-UTR m6A. (Sun H, et al., 2021)

Features of m6Am-Exo-seq

m6Am-Exo-seq is a specialized tool that provides unprecedented specificity for cap-associated methylation analysis, overcoming the principal limitation of antibody cross-reactivity in standard m6A profiling.

Core Technological Advantages

High Specificity for Cap m6Am: The exonuclease pretreatment step selectively removes non-capped RNA fragments, ensuring targeted analysis of true transcription start site (TSS)-proximal m6Am.
Reduced Background Noise: By eliminating internal m6A signals, the protocol significantly lowers background interference, enhancing signal-to-noise ratio and data clarity.
Functional Insights into Cap Biology: Enables direct investigation of the relationship between m6Am methylation, cap-binding complexes, and downstream mRNA fate decisions.
Compatibility with Low-Abundance Transcripts: The enrichment strategy allows for effective profiling even for lowly expressed transcripts that harbor this modification.

Our Services

By combining optimized enzymatic specificity with advanced immunoprecipitation, CD BioSciences delivers comprehensive and reliable m6Am-Exo-seq services to precisely delineate the landscape of cap-proximal m6Am in your biological systems. Utilizing rigorously validated protocols and antibodies, we generate high-fidelity, modification-specific data that empower researchers to accurately investigate how m6Am dynamics regulate mRNA stability and translation, offering critical insights into development, disease mechanisms, and therapeutic targeting.

Workflow of m6Am-Exo-seq Service

Our m6Am-Exo-seq service follows a rigorously optimized two-step enrichment pipeline, designed to isolate and sequence cap-protected RNA fragments with high specificity. This integrated workflow combines enzymatic precision with immunoprecipitation-based capture to ensure accurate identification of genuine m6Am modifications while effectively excluding internal m6A background signals. Each step is subjected to stringent quality control to guarantee reproducibility and data integrity for downstream analysis.

Cap-Protected RNA Enrichment

Total RNA undergoes treatment with a 5'→3' exonuclease (such as XRN-1) to selectively degrade uncapped RNA species, thereby enriching for intact, capped transcripts that may contain m6Am modifications.

RNA Fragmentation & Quality Control

The enriched RNA pool is fragmented to optimal sizes for sequencing library construction, followed by rigorous quality assessment to verify fragment size distribution and RNA integrity.

Immunoprecipitation

Fragmented RNA is incubated with highly validated antibodies specific to m6A/m6Am epitopes. Multiple wash steps ensure specific capture of methylated RNA fragments while minimizing non-specific binding.

Library Preparation & Sequencing

The immunoprecipitated RNA undergoes end repair, adapter ligation, reverse transcription, and PCR amplification to generate sequencing-ready libraries. Libraries are then sequenced on Illumina platforms with appropriate depth to ensure comprehensive coverage.

Bioinformatics Analysis Pipeline

Raw sequencing reads are processed through our customized analysis pipeline including: adapter trimming, alignment to reference genome, m6Am peak calling with background correction, motif analysis, transcript annotation, and quantification of modification levels.

Data Delivery & Technical Support

We provide all raw sequencing data, processed results files, detailed analytical reports with visualization, and dedicated scientific consultation to support your data interpretation and publication needs.

Compatible Samples & Research Applications

CD BioSciences designs its m6Am-Exo-seq service with exceptional versatility, supporting a diverse range of biological materials and enabling cutting-edge research across multiple scientific frontiers. Through optimized protocols and rigorous quality control, we ensure reliable performance with various sample types, while generating data that directly addresses fundamental questions in epitranscriptomic regulation, disease mechanisms, and therapeutic development.

Supported Sample Types

Cell Lines & Primary Cells: Including adherent, suspension, stem, and differentiated cells.
Animal & Clinical Tissues: Fresh, snap-frozen, or RNAlater-preserved samples from model organisms and human specimens.
Low-Input & Specialized Samples: Optimized protocols for limited materials such as sorted cell populations, tissue biopsies, and microdissected samples.
Plant & Microbial Samples: Customized approaches for plant tissues and selected microorganisms (subject to consultation).

Primary Research Applications

Cap Biology & Translation Regulation: Study how m6Am influences cap-dependent translation initiation and mRNA stability.
Disease Mechanism Studies: Investigate m6Am dysregulation in cancer, neurological disorders, and metabolic diseases.
Development & Differentiation: Profile dynamic m6Am changes during embryonic development and cell lineage specification.
Viral-Host Interactions: Analyze viral RNA cap modifications and their role in infection and immune evasion.
Therapeutic Response Monitoring: Track m6Am landscape alterations in response to drug treatments or targeted therapies.

Our Advantages

Unmatched m6Am Specificity: Our optimized workflow is specifically designed to isolate the cap-associated m6Am signal, providing data you can trust.
Expertise in Cap Biology: Our team possesses deep knowledge of RNA cap modifications and their analysis, ensuring proper experimental design and data interpretation.
Integrated Analytical Pipeline: We provide more than peak calling; our analysis includes TSS alignment, motif validation, correlation with RNA expression/stability data, and comparative analysis across conditions.
End-to-End Project Support: Dedicated scientific support guides your project from initial consultation through final data interpretation.

Accurately profiling the m6Am modification is key to understanding its unique role in mRNA cap biology and gene regulation. CD BioSciences' m6Am-Exo-seq service provides the targeted methodology and analytical precision required for these focused investigations. To offer a complete solution for RNA cap modification analysis, we also support complementary techniques such as LC-MS/MS for absolute quantification and m6A-seq for global internal methylation profiling. This allows us to provide a fully customized, multi-faceted strategy for your epitranscriptomic research. Contact us today to discuss your project and receive a detailed proposal.

Reference

1. Sun H, Li K, Zhang X, et al. m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome[J]. Nature Communications, 2021, 12(1): 4778.