caRNA Modification Sequencing Services for Epitranscriptomics

Technology Principle & Advantages

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.

1. 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.
2. 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.
3. caRNA Fragmentation. Extracted caRNA is randomly fragmented to improve immunoprecipitation resolution. QC: Fragment size distribution checked by capillary electrophoresis.
4. 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.
5. 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.
6. 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.

Bioinformatics Analysis Pipeline

The standard analysis pipeline covers raw data processing through functional enrichment. Each module produces publication-ready outputs.

1. Raw data processing. Adapter trimming, low-quality read removal, read length filtering. Q30 scores and sequencing statistics reported.
2. Read alignment. Clean reads aligned to the reference genome.
3. 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.
4. 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).
5. 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.
6. Motif analysis. Sequence motifs enriched at modification sites are identified, revealing sequence preferences of the modification machinery.
7. 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.

Data Deliverables

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.

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.