caRNA Modification Sequencing Services for Epitranscriptomics
caRNA modification sequencing combines subcellular chromatin fractionation with modification-specific immunoprecipitation (MeRIP-Seq) to profile covalent RNA modifications — including m6A, m5C, m1A, Ψ, m7G, 2′-O-Me, ac4C, and m6Am — specifically on chromatin-associated transcripts. By enriching chromatin-associated RNA before modification detection, the approach targets modifications most likely to affect local chromatin state and transcriptional control, avoiding the dilution of signal by abundant cytoplasmic transcripts. CD Genomics provides end-to-end support from cell fractionation through sequencing and bioinformatics analysis.
Key Highlights of Our caRNA Modification Sequencing Service:
Chromatin-Specific Enrichment: Cell fractionation isolates chromatin-associated RNA before modification IP, focusing on modifications relevant to chromatin state and local transcriptional control.
Dual Spike-In Controls: Positive and negative synthetic reference RNAs enable IP efficiency monitoring and quantitative comparison across samples — a feature not standard in conventional MeRIP-seq.
Multi-Modification Coverage: Supports profiling of 8 RNA modification types on a unified workflow with the same chromatin enrichment and IP framework.
caRNA-Type Resolution: Modification sites annotated by caRNA category (eRNA, asRNA, paRNA, repeat RNA, cenRNA) and coding capacity, revealing modification patterns unique to specific regulatory RNA classes.
Chromatin-associated RNA (caRNA) encompasses a diverse group of RNA molecules that interact directly or indirectly with chromatin, including enhancer RNAs (eRNAs), promoter-associated RNAs (paRNAs), antisense RNAs (asRNAs), repeat RNAs (including centromeric RNAs), and nascent pre-mRNAs. These RNA species participate in transcriptional regulation, chromatin remodeling, and the maintenance of genome organization.
caRNA modification sequencing combines subcellular fractionation with RNA modification-specific immunoprecipitation to profile covalent RNA modifications specifically on chromatin-associated transcripts. The approach first isolates chromatin-enriched fractions from cells or tissues, extracts associated RNA, and then applies antibody-based enrichment — built on the MeRIP-seq principle — to capture RNA fragments carrying modifications such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ). High-throughput sequencing and computational analysis then map modification positions across caRNA subtypes at transcriptome scale.
Unlike standard MeRIP-seq, which profiles modifications across total RNA, caRNA modification sequencing targets the chromatin-associated compartment, providing a focused view of modifications that directly influence chromatin state and local transcriptional control.
Technology Principle & Advantages
Principle
The method proceeds through three sequential stages:
1Chromatin Enrichment
Intact cells are lysed and nuclei are isolated by centrifugation through a sucrose cushion. The nuclear fraction is further processed to separate chromatin-associated components from nucleoplasmic material, yielding a chromatin-enriched fraction containing caRNA.
2Modification-Specific IP
caRNA is extracted and randomly fragmented. A portion is retained as the input control. The remainder is incubated with a modification-specific antibody (e.g., anti-m6A) to enrich fragments carrying the target modification. Two classes of synthetic Spike-In RNA — one carrying the modification and one lacking it — are added before IP to serve as internal references for enrichment efficiency and technical bias correction.
3Sequencing & Analysis
Both IP and input libraries are sequenced. Reads are aligned to the reference genome. Enriched regions (peaks) are identified by comparing IP signal against input. Modification sites are annotated to caRNA types and genomic features. Differential modification analysis between sample groups identifies condition-dependent changes.
Advantages
Targeted Chromatin Compartment Analysis
By enriching chromatin-associated RNA before modification detection, the approach focuses on modifications most likely to affect local chromatin state and transcription, avoiding the dilution of signal by abundant cytoplasmic transcripts.
Spike-In Quantitative Framework
The dual-control design — using both a modified and an unmodified synthetic reference RNA — provides internal normalization for IP efficiency. This allows more reliable cross-sample comparison than conventional MeRIP-seq, which typically lacks internal quantification standards.
Single-Platform Multi-Modification Coverage
The workflow supports profiling of m6A, m5C, m1A, Ψ, m7G, 2′-O-methylation, ac4C, and m6Am through the same chromatin enrichment and immunoprecipitation framework with appropriate antibodies.
caRNA-Type Annotation
Bioinformatics analysis classifies each modification site by its host caRNA category (eRNA, asRNA, paRNA, repeat RNA, cenRNA, nascent pre-mRNA), revealing modification patterns unique to specific regulatory RNA classes.
Modification Types
Supported Modification Types
Modification
Full Name
Detection Method
m6A
N6-methyladenosine
Anti-m6A antibody IP
m5C
5-methylcytosine
Anti-m5C antibody IP
m1A
N1-methyladenosine
Anti-m1A antibody IP
Ψ
Pseudouridine
Anti-Ψ antibody IP
m7G
N7-methylguanosine
Anti-m7G antibody IP
2′-O-Me
2′-O-methylation
Anti-2′-O-Me antibody IP
ac4C
N4-acetylcytidine
Anti-ac4C antibody IP
m6Am
N6,2′-O-dimethyladenosine
Anti-m6Am antibody IP
Additional modification types may be accommodated upon consultation. Each modification type requires its own IP and input library pair.
Workflow
Experimental Workflow
The caRNA modification sequencing workflow spans six stages, each with integrated quality control checkpoints. The complete process — from nuclear enrichment to bioinformatics analysis — is outlined below.
Nuclear Enrichment. Harvested cells are washed with cold PBS/EDTA, lysed, and the lysate is layered over a sucrose cushion. High-speed centrifugation separates the cytoplasmic fraction (supernatant) from the nuclear pellet. QC: Assess nuclear purity by microscopy; verify chromatin fraction protein content.
Chromatin Fraction Isolation. The nuclear pellet is washed, resuspended in glycerol buffer, and treated with nuclear lysis buffer to disrupt the nuclear membrane. Centrifugation yields a chromatin-enriched fraction containing caRNA. QC: Fraction purity monitored by protein markers.
caRNA Fragmentation. Extracted caRNA is randomly fragmented to improve immunoprecipitation resolution. QC: Fragment size distribution checked by capillary electrophoresis.
Spike-In Addition and Immunoprecipitation. Synthetic Spike-In controls (positive: carries the target modification; negative: lacks it) are added to the fragmented caRNA. Modification-specific antibody is used to capture modified fragments (IP). A parallel aliquot is retained as the input control. QC: IP efficiency monitored by qPCR against Spike-In references; input sample preserved for peak calling.
Library Preparation and Sequencing. IP and input RNA are separately converted to cDNA libraries, amplified, and sequenced on an Illumina platform (paired-end mode recommended). QC: Library size distribution, Q30 scores, and duplication rate assessed.
Bioinformatics Analysis. Reads are aligned to the reference genome. Peak calling (MACS) identifies enriched regions in IP versus input. Modification sites are annotated to caRNA types and genomic features. Differential modification analysis (diffReps) compares modification levels between experimental groups. Downstream analyses include GO/KEGG enrichment, motif discovery, and metagene profiling.
Sample
Sample Requirements
Sample quality and quantity directly affect chromatin enrichment efficiency, IP specificity, and modification detection sensitivity. Recommended input amounts and storage conditions are summarized below.
Sample Type
Recommended Amount
Notes
Cultured cells
≥ 1 × 10⁸
Harvested fresh; programmed freezing for live-cell preservation to maintain nuclear integrity
Tissue
≥ 400 mg
Applicable to heart, liver, spleen, lung, kidney, and common tissues; contact us for specialized tissue types
Purified caRNA
≥ 4 µg
Extracted from chromatin-enriched fraction; concentration ≥ 50 ng/µL recommended
Shipping and storage:
Samples should be placed in 1.5 mL or cryovials, sealed with parafilm, and shipped on dry ice.
Live cells require programmed freezing for cryopreservation to maintain nuclear integrity for subsequent fractionation.
Tissue samples may be treated with TRIzol or RNA stabilization reagent, snap-frozen in liquid nitrogen, and stored at −80°C.
Purified caRNA should be dissolved in nuclease-free water and stored at −80°C; avoid repeated freeze-thaw cycles.
Bioinformatics
Bioinformatics Analysis Pipeline
The standard analysis pipeline covers raw data processing through functional enrichment. Each module produces publication-ready outputs.
Raw data processing. Adapter trimming, low-quality read removal, read length filtering. Q30 scores and sequencing statistics reported.
Read alignment. Clean reads aligned to the reference genome.
Peak calling and annotation. MACS identifies modification-enriched regions (peaks) by comparing IP against input. Each peak is annotated to its host caRNA type — non-coding (lncRNA, circRNA, snoRNA, snRNA) or coding (pre-mRNA) — and further classified into caRNA categories (eRNA, asRNA, paRNA, repeat RNA, cenRNA) based on genomic context.
Differential modification analysis. diffReps compares modification levels between sample groups, identifying significantly differentially modified regions based on fold change and statistical significance (P-value calculation requires ≥ 3 biological replicates per group).
GO and KEGG enrichment. Differentially modified pre-mRNA host genes and non-coding caRNA neighboring genes are analyzed separately for Gene Ontology (molecular function, cellular component, biological process) and KEGG pathway enrichment. The cis-regulatory analysis of neighboring genes for non-coding caRNAs provides insight into local transcriptional regulatory mechanisms.
Motif analysis. Sequence motifs enriched at modification sites are identified, revealing sequence preferences of the modification machinery.
Classification and distribution. Pie charts show the distribution of modification sites across caRNA categories. Metagene profiles display modification density along transcript features.
Optional advanced analyses — including integration with RNA-seq expression data, comparison with chromatin state data (ChIP-seq, ATAC-seq), and multi-modification co-occurrence analysis — are available upon request.
Demo
Demo Results
The following representative visualizations illustrate the types of results delivered with each caRNA modification sequencing project. All panels are labeled "Representative data."
Representative caRNA modification sequencing analysis outputs delivered with each project. (A) Peak calling output showing enrichment of modification signal (IP vs. input) with caRNA-type annotation. (B) Pie chart displaying modification site distribution across caRNA categories (eRNA, asRNA, paRNA, repeat RNA, cenRNA). (C) Volcano plot highlighting significantly hyper- and hypo-modified caRNAs between experimental groups. (D) GO and KEGG enrichment bar charts for genes associated with differentially modified caRNAs. (E) Sequence logo of enriched nucleotide motif at modification sites. (F) Metagene plot showing modification signal distribution along gene body coordinates. (G) Venn diagram of modification site overlap across multiple samples or conditions.
Modification-enriched regions identified by MACS with coordinates and enrichment scores
caRNA annotation table
Classification of each modification site by caRNA type and genomic feature
Differential modification results
Significantly changed modification sites between conditions, with fold change and P-values
GO/KEGG enrichment report
Functional enrichment analysis for genes associated with differentially modified caRNAs
Motif analysis results
Enriched sequence motifs at modification sites with significance scores
Visualization package
Classification pie charts, volcano plots, metagene profiles, Venn diagrams, and heatmaps
Methods and analysis report
Detailed description of all data processing and analysis steps
Applications
Applications
caRNA modification sequencing addresses a central question in epitranscriptomics: how do chemical modifications on chromatin-associated transcripts influence local chromatin state, transcriptional regulation, and gene expression programs?
Cancer Epigenetics
caRNA modification profiling reveals how aberrant m6A deposition on chromatin-associated transcripts — such as centromeric RNA — contributes to oncogenic transcriptional programs. m6A-modified cenRNA stabilizes centromeric protein localization and maintains chromosome segregation fidelity in cancer cells, identifying caRNA modification as a potential layer of cancer vulnerability.
Transcriptional Regulation
Modifications on eRNAs and paRNAs modulate the stability and activity of these regulatory RNAs at their sites of transcription. m6A on caRNAs can recruit reader proteins that influence chromatin state and RNA polymerase II dynamics, linking epitranscriptomic marks directly to transcriptional output.
Developmental Biology
Dynamic caRNA modification patterns during cell differentiation and early embryonic development regulate gene expression programs. Profiling modifications in chromatin-associated transcripts provides insight into how epitranscriptomic marks shape cell fate decisions.
Disease Biomarker Discovery
Tissue-specific and disease-associated caRNA modification signatures may serve as biomarkers. Differential modification analysis between healthy and disease states identifies caRNA modification events with potential diagnostic or prognostic utility.
For complementary epitranscriptomic analyses, MeRIP-Seq provides transcriptome-wide modification profiling from total RNA, and DRIPc-seq enables R-loop detection — another chromatin-associated nucleic acid feature relevant to transcriptional regulation.
Method Comparison
Method Comparison: caRNA Modification Sequencing vs. Standard MeRIP-Seq
caRNA modification sequencing and standard MeRIP-seq share the same modification-specific immunoprecipitation principle but differ fundamentally in starting material, quantification framework, and the depth of chromatin-level annotation.
Feature
caRNA Modification Sequencing
Standard MeRIP-Seq
Starting material
Chromatin-enriched fraction
Total RNA
RNA population profiled
Chromatin-associated RNA specifically
All cellular RNA (predominantly cytoplasmic)
Chromatin state relevance
Direct — modifications mapped on chromatin-interacting transcripts
Indirect — chromatin-associated signal diluted by cytoplasmic background
Included — neighboring gene analysis for non-coding caRNAs
Not standard
Sensitivity to chromatin-localized modification changes
High
Low due to cytoplasmic RNA dominance
Suitable for
Studies focused on chromatin-level epitranscriptomic regulation
Broad transcriptome-wide modification surveys
Selection guidance:
Choose caRNA modification sequencing when your research question concerns how RNA modifications influence chromatin state, local transcription, or the function of specific chromatin-associated RNA species (eRNAs, paRNAs, cenRNAs).
Choose standard MeRIP-Seq when you need a broad survey of RNA modifications across the total transcriptome without subcellular fractionation.
For studies of RNA-DNA hybrid structures at chromatin, DRIPc-seq and ssDRIP-Seq provide complementary R-loop detection.
