GLORI-seq: The Gold Standard for Single-Base m6A Quantification

Unlock the potential of m6A research with GLORI-seq, the most precise and accurate method for profiling m6A modifications at single-base resolution. GLORI-seq offers absolute quantification of m6A modifications across the transcriptome, without the biases associated with traditional antibody-dependent techniques. Whether you're exploring RNA dynamics in cancer, developmental biology, or stress responses, GLORI-seq is your go-to solution for reliable, high-resolution m6A analysis.

Key Benefits:

Single-Base Resolution: Achieve unparalleled detail in your m6A data.
Absolute Quantification: Accurately measure m6A levels across diverse samples.
High Sensitivity: Detect even low-abundance modifications in transcripts.

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GLORI-seq workflow diagram illustrating four steps: RNA extraction, fragmentation and chemical treatment with glyoxal and nitrite, library preparation, and data analysis, highlighting single-base m6A quantification with high resolution.

Introduction

Methodology: From Sample to Insight

GLORI-seq offers a streamlined, reliable approach for studying m6A modifications in RNA. Here's a breakdown of the methodology that ensures high-quality, precise data:

1. RNA Extraction and Fragmentation

High-quality RNA is extracted from your sample and fragmented to an optimal size for sequencing. This ensures the resulting data is accurate and represents the full spectrum of RNA modifications.

2. Chemical Treatment: Targeting Unmethylated Adenosines

The core innovation of GLORI-seq is the selective chemical deamination of unmethylated adenosines to inosines using glyoxal and nitrite. m6A-modified adenosines remain unchanged, allowing for accurate detection of m6A modifications while excluding other modifications.

3. Library Preparation

The modified RNA is reverse transcribed to create complementary DNA (cDNA). Sequencing libraries are then prepared using standard protocols, providing the necessary material for high-throughput sequencing.

4. Sequencing and Data Acquisition

Sequencing is performed on standard platforms, like Illumina NovaSeq, to generate high-resolution data. The resulting sequencing reads capture A-to-G conversion sites that signal m6A modifications across the transcriptome.

5. Bioinformatics Analysis

Once sequencing is complete, bioinformatics tools are used to:

Perform quality control on the raw data.
Align reads to the reference genome, identifying A-to-G conversion sites.
Quantify m6A modifications at each position.
Visualize the data through heatmaps and distribution plots.
Analyze the functional significance of m6A modifications in gene regulation and biological pathways.

GLORI-seq methodology flowchart showing five key steps: RNA extraction and fragmentation, selective chemical treatment of unmethylated adenosines, library preparation, high-throughput sequencing, and bioinformatics analysis to identify and quantify m6A modifications at single-base resolution.

Efficient and Accurate Research

GLORI-seq's efficient workflow ensures high-quality, reproducible data, allowing you to focus on biological insights. The process seamlessly combines chemical precision, sequencing, and bioinformatics for reliable m6A profiling, advancing your research in RNA regulation.

Advantages

Technical Innovation: Chemical Precision

A three-step schematic of m6A detection chemistry showing: (1) guanosine (G) protection, (2) adenosine (A) deamination to inosine (I), and (3) guanosine deprotection. Includes labeled RNA strands with m6A, G⁺ (N¹,N²-dihydroxyguanosine), and inosine, along with a legend clarifying each modification.

GLORI-seq transforms how we analyze m6A modifications by moving away from traditional antibody-based methods. Instead, it uses a cutting-edge chemical process, ensuring greater accuracy and fewer biases.

Chemical Reaction for Unbiased Detection: Using glyoxal and nitrite, GLORI-seq selectively deaminates unmethylated adenosines to inosines. This ensures that m6A modifications remain untouched, while non-methylated sites are chemically modified—allowing researchers to differentiate between them with ease.
Precise Identification of m6A Sites: This deamination leads to a unique A-to-G conversion during sequencing, offering a clean, clear signal for m6A sites. No more interference from other RNA modifications—just highly accurate, single-base resolution data.
High Sensitivity and Specificity: GLORI-seq achieves an exceptional A-to-I conversion rate of over 98%, making it possible to detect even rare m6A sites. Other bases barely undergo any conversion, ensuring the integrity of your data.
Unbiased Profiling: Unlike antibody-based methods, GLORI-seq doesn't rely on specific sequence motifs. This means it captures the full diversity of m6A modifications across your transcriptome, without any biases creeping in.

With its combination of chemical precision, high specificity, and reliable results, GLORI-seq is a game-changer for any researcher looking to explore the complex world of RNA modifications with unmatched clarity.

mA proportion = A/(A+G)

The principle of GLORI-seq technology

Quantitative Accuracy: Absolute m6A Profiling

GLORI-seq not only revolutionizes how m6A modifications are detected but also takes the quantification of these modifications to a new level of precision. By calculating the exact proportion of A-to-G conversions at each position, GLORI-seq offers absolute quantification of m6A, making it the most reliable tool for m6A profiling across the transcriptome.

Why does this matter?

Unbiased Detection
Unlike traditional methods, GLORI-seq eliminates the reliance on sequence motifs or antibody-based enrichments that could skew data. The chemical reaction used in GLORI-seq ensures that m6A modifications are mapped comprehensively, without any bias towards specific sequences or secondary structures. This allows for a full transcriptome analysis, ensuring no m6A sites are overlooked.
Increased Sensitivity for Low-Abundance m6A Sites
GLORI-seq is highly sensitive, allowing you to detect m6A modifications in low-abundance transcripts that other methods might miss. Whether you're studying rare cell types or low-expressed genes, GLORI-seq guarantees accurate profiling of even the smallest modifications, providing a more complete picture of m6A's role in gene regulation.
Single-Base Resolution
The precision of GLORI-seq goes beyond just detecting m6A—it offers single-base resolution, allowing you to pinpoint exactly where each modification occurs in the RNA sequence. This level of detail is essential for understanding the functional impact of m6A on gene expression, splicing, and translation.
Seamless Integration with Sequencing Platforms
Compatible with widely-used sequencing platforms like Illumina NovaSeq, GLORI-seq fits seamlessly into your existing research workflow.

The Impact on Your Research

With GLORI-seq, you're not just detecting m6A—you're measuring it with unmatched precision. This allows you to explore the dynamics of m6A modifications in depth, gaining insights into how these modifications influence cellular processes, gene expression, and disease progression. Whether studying developmental biology, neurological disorders, or cancer, the ability to quantify m6A at single-base resolution opens new doors for discovering RNA regulation mechanisms and therapeutic targets.

Advantages Over Traditional Methods

GLORI-seq offers several key advantages over traditional m6A sequencing methods, enhancing the accuracy, sensitivity, and comprehensiveness of m6A profiling. The following table summarizes the benefits:

Feature	GLORI-seq	Antibody-Based Methods	Enzyme-Based Methods
Resolution	Single-base resolution, high precision	Low resolution, limited by sequence motifs	Moderate resolution, dependent on enzyme specificity
Quantification	Absolute quantification of m6A levels	Relative quantification	Relative quantification
Bias Toward Motifs	No reliance on sequence motifs	Bias towards specific sequence motifs (e.g., ACA)	Bias towards specific recognition sites (e.g., ACA)
Sensitivity	High sensitivity, detects low-abundance m6A	Lower sensitivity, may miss low-abundance sites	Moderate sensitivity, but still limited by enzyme's activity
RNA Input Requirement	Low RNA input requirement	High RNA input required	Moderate RNA input requirement
Compatibility with Platforms	Compatible with standard sequencing platforms (e.g., Illumina NovaSeq)	Requires specialized platforms or antibodies	Limited compatibility with sequencing platforms
Versatility	Unbiased detection across the transcriptome	Limited to regions with specific motifs	Limited to enzyme-specific regions

Applications

Applications: Advancing Epitranscriptomics

GLORI-seq provides a transformative tool for studying m6A modifications, offering researchers unparalleled insights across several critical biological fields. Here's how it is advancing research:

1. Developmental Biology: Understanding m6A's Role in Cell Differentiation

GLORI-seq enables precise tracking of m6A modifications during embryogenesis and tissue differentiation, providing valuable insights into how RNA modifications regulate cell fate and organ development. By mapping m6A at key developmental stages, researchers can uncover new regulatory pathways that are essential for normal growth and could help understand how developmental disorders arise at the molecular level.

2. Neuroscience: Decoding m6A in Neuronal Function

m6A modifications play a critical role in processes like synaptic plasticity and neuronal signaling. GLORI-seq allows researchers to map these modifications in the brain with high resolution, shedding light on their influence in brain function, learning, memory, and complex behaviors. This technique also opens the door to understanding how m6A modifications impact neurodegenerative diseases and neurodevelopmental disorders.

3. Cancer Research: m6A's Role in Tumor Progression

In cancer, m6A is involved in regulating the genes that control tumor growth, metastasis, and chemoresistance. By providing a detailed analysis of m6A modifications in cancer cells, GLORI-seq helps identify how these modifications affect tumorigenesis and could uncover molecular signatures involved in cancer progression. This deeper understanding paves the way for novel therapeutic strategies targeting m6A-modified genes.

4. Stress Response: m6A Modifications Under Environmental Stress

Understanding how cells react to stress is key to many biological research areas, including stress adaptation, cell survival, and immune function. GLORI-seq allows researchers to profile how environmental factors like hypoxia or heat shock induce specific m6A modifications. This data is crucial for exploring how m6A mediates responses to stress, providing insights into how organisms adapt to external stresses in fields ranging from plant biology to immunology.

5. RNA Regulation: Exploring m6A's Global Role

Beyond specific diseases or conditions, m6A plays a widespread role in regulating RNA processing and gene expression across various biological systems. GLORI-seq is an essential tool for understanding the broader influence of m6A on RNA stability, splicing, and translation. Researchers can use GLORI-seq to examine global RNA modifications and how they drive important cellular processes across diverse contexts, from cellular homeostasis to response to environmental cues.

Service Workflow

Service Workflow: From Inquiry to Data Delivery

GLORI-seq is designed to integrate seamlessly into your research process. Our service workflow ensures that from the initial consultation to the delivery of data, you receive clear communication, high-quality results, and an efficient experience. Here's how it works:

Bioinformatics

Bioinformatics Analysis: Turning Data into Insights

Once you have your GLORI-seq data, the next step is to unlock its potential through bioinformatics analysis. Our comprehensive analysis pipeline transforms sequencing data into meaningful, actionable insights. Here's how we help you interpret the results:

1. Quality Control: Ensuring High-Quality Data

We begin by performing quality control (QC) to ensure the integrity of your sequencing data. This step helps us identify and eliminate any potential issues—such as low-quality reads or sequencing biases—ensuring that your final dataset is clean and reliable. This guarantees that the insights you derive are based on accurate, high-quality data.

2. Alignment and Mapping: Pinpointing m6A Modifications

Once the data passes QC, we map the sequencing reads to a reference genome. This step allows us to accurately locate m6A modification sites by identifying A-to-G conversion points. The result is a precise map of m6A sites across the transcriptome, offering clarity on the specific locations and potential effects of m6A modifications in your samples.

3. Quantification: Measuring m6A Levels

With GLORI-seq, we go beyond simple detection. We calculate the exact proportion of A-to-G conversions at each m6A site, providing absolute quantification of m6A modification levels. This enables you to track changes in m6A abundance across different conditions or experimental treatments, allowing for detailed insights into how m6A modifications correlate with gene expression and regulatory processes.

4. Visualization: Simplifying Complex Data

Interpreting high-throughput data can be overwhelming, but our visualization tools make it easy. Using heatmaps, distribution plots, and other graphical representations, we present your data in an intuitive format. These visual tools allow you to quickly identify patterns, anomalies, and key trends, such as differences in m6A modifications between conditions or across samples.

5. Functional Analysis: Linking m6A to Biological Pathways

The final step of our analysis focuses on the biological significance of m6A modifications. By integrating your m6A data with gene annotations, we identify regulated pathways and gene networks that are influenced by m6A. This helps connect your m6A findings to biological functions like gene expression, RNA stability, splicing, and cellular stress responses, offering deeper insights into the regulatory role of m6A in your system.

Vertical infographic showing five key steps of GLORI-seq bioinformatics analysis: Quality Control (filtering low-quality reads), Alignment & Mapping (locating A-to-G sites), Quantification (measuring A-to-G conversions at m6A sites), Visualization (displaying data with heatmaps and plots), and Functional Analysis (linking m6A sites to biological pathways).

Sample Requirements

Sample Requirements: Ensuring Optimal Results

To achieve accurate and reliable GLORI-seq results, it is crucial to submit high-quality samples. Below are the sample guidelines to ensure optimal performance:

Sample Types

Cell Lines
Tissues
Body Fluids (Blood, Plasma, Serum, Cerebrospinal Fluid, Urine)
RNA Samples: For other types, please inquire for further guidance.

Sample Quantity

Cells: Minimum 2×10⁷ cells
Tissue: 50 mg
Body Fluids:
- Blood, Serum, Plasma: 2-3 ml
- Cerebrospinal Fluid: 5 ml
- Urine: 50 ml
Total RNA: Minimum 2 µg
- RNA Quality: OD260/280 ≥ 1.8, OD260/230 ≥ 1.5
- Electrophoresis: Clear, intact RNA bands with no degradation.

Sample Storage and Shipping

Storage:
- RNA: Store at -80°C in RNase-free tubes.
- Cells/Tissues: Use TRIzol or RNA stabilizer and freeze immediately in liquid nitrogen or store at -80°C.
- Blood Samples: Use EDTA as anticoagulant.
Shipping:
- Ship samples with dry ice in 1.5 mL tubes with sealed caps.
- Ensure proper packaging to maintain sample integrity during transport.

FAQ