5caC DIP-Seq Service: Genome-wide Mapping of Active DNA Demethylation
Dynamic DNA demethylation is hard to capture: 5caC is rare, transient, and highly context-specific. Our 5caC DIP-Seq service delivers sensitive, genome-wide 5-carboxylcytosine profiles to decode TET/TDG-dependent regulation.
- Optimized 5caC DIP-Seq (5caC-specific IP + high-throughput sequencing): higher sensitivity for rare 5caC; robust genome-wide maps of active DNA demethylation
- Genome-wide 5caC peaks at promoters, enhancers, and repeats: direct TET/TDG pathway readout; prioritized regulatory regions for follow-up ChIP-seq / RNA-seq
- Integrated wet-lab + bioinformatics pipeline for 5caC DIP-Seq: less assay optimization and analysis overhead; samples-to–publication-ready 5caC data in a single service
5caC DIP-Seq Overview: What This Assay Measures and Why It Matters
What is 5caC and why does it matter?
In mammals, active DNA demethylation is mediated by TET enzymes that sequentially oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are typically low-abundance and short-lived, because they are removed by thymine DNA glycosylase (TDG) and base-excision repair. This transience means 5caC marks genomic regions undergoing active methylation turnover, making it a sensitive readout of dynamic regulatory processes.
TET–TDG-mediated oxidation pathway from 5mC to 5caC
What is 5caC DIP-Seq?
5caC DIP-Seq is a genome-wide assay that profiles 5-carboxylcytosine in DNA by combining:
- DNA immunoprecipitation (DIP) with a highly specific anti-5caC antibody to enrich 5caC-containing DNA fragments, and
- high-throughput sequencing to map these fragments back to the reference genome.
The result is a genome-wide 5caC modification landscape that can be directly compared between cell types, tissues, or experimental conditions.
Why profile 5caC?
- 5caC is a late oxidation intermediate that accumulates where active DNA demethylation is engaged, particularly at regulatory elements under strong TET/TDG activity.
- Genome-wide 5caC profiling can reveal:
- Dynamic turnover at enhancers and promoters
- TET/TDG pathway activity in development and reprogramming
- How DNA demethylation reshapes gene regulatory networks
Our 5caC DIP-Seq platform translates these mechanistic insights into robust, interpretable datasets for your epigenetics projects.
Key Research Applications of 5caC DIP-Seq in Active DNA Demethylation
Our 5caC DIP-Seq service is designed for research groups that need:
- TET/TDG pathway interrogation
- Map how TET or TDG loss-of-function, catalytic mutants, or pharmacologic perturbations redistribute 5caC.
- Developmental and stem cell epigenetics
- Track 5caC accumulation during lineage specification in embryonic or induced pluripotent stem cells to pinpoint enhancers undergoing active demethylation.
- Chromatin and transcription factor biology
- Overlay 5caC peaks with ChIP-seq of transcription factors or architectural proteins to test whether 5caC facilitates or restricts binding at suboptimal motifs.
- Multi-mark oxidation profiling
- Combine 5caC DIP-Seq with 5hmC DIP-Seq, 5fC DIP-Seq, or base-resolution methods to reconstruct the full 5mC oxidation trajectory at key genes.
Service portfolio: single- and multi-mark assays
We offer 5caC DIP-Seq:
- As a standalone assay focused on 5caC enrichment and mapping.
- In bundled oxidation panels with 5hmC DIP-Seq and 5fC DIP-Seq for comprehensive pathway analysis.
- Integrated with WGBS, ATAC-seq, ChIP-seq, or RNA-seq data (provided by you or run through our other services) to build multi-omic regulatory models.
Technical Principles: How 5caC DIP-Seq Enriches and Maps 5-Carboxylcytosine
The core of 5caC DIP-Seq is a high-affinity antibody that selectively recognizes 5caC-modified cytosines in genomic DNA.
Key features of our IP chemistry:
- Fragmented genomic DNA (~100–300 bp) is denatured and incubated with anti-5caC antibody.
- Immune complexes are captured on protein A/G beads, washed under stringent conditions, and eluted.
- A parallel input control (non-immunoprecipitated, fragmented DNA) is processed identically from library preparation onward to model background.
Enriched and input fractions are converted to Illumina-compatible libraries and sequenced to generate millions of reads. After alignment to the reference genome:
- Enrichment over input is quantified to identify 5caC peaks.
- Peaks are associated with genomic features (promoters, exons, introns, intergenic regions, repeats) and genes.
- Signal can be aggregated around transcription start sites, enhancers, or other regions of interest.
For comparative projects (e.g., wild-type vs. TET/TDG perturbation, differentiation time course):
- Normalized coverage at each peak is used to compute differential 5caC peaks.
- Peaks are further linked to genes for functional enrichment analysis and integrated with gene expression or chromatin accessibility data when available.
This principled analysis framework ensures that 5caC DIP-Seq yields both locus-level and systems-level insights.
Why Choose Our 5caC DIP-Seq Service: Sensitivity, QC, and Expert Support
We provide a complete workflow:
- Sample QC and genomic DNA extraction (if starting from cells, tissues, or blood)
- Optimized 5caC DIP enrichment
- Library construction and high-throughput sequencing
- Bioinformatics analysis and visualization
You receive ready-to-interpret results without having to troubleshoot immunoprecipitation chemistry, sequencing, or downstream pipelines.
5caC is extremely rare, so enrichment efficiency and specificity are critical.
Our workflow includes:
- Validated 5caC antibody performance and binding conditions
- IP efficiency assessment by qPCR at positive/negative control loci (where available)
- Library QC (size distribution, concentration, adapter dimer removal)
- Sequencing QC (yield, Q30, mapping rate, duplication rate)
Only datasets passing predefined QC thresholds proceed to full analysis.
Our bioinformatics team is experienced in processing DIP-based datasets and integrating them with ChIP-seq, ATAC-seq, RNA-seq, and DNA methylation data. Deliverables are designed to be directly usable in manuscripts, grant figures, and presentations.
5caC DIP-Seq Workflow: From Sample Submission to Genome-wide 5caC Maps
- DNA extraction and QC: Isolate high-quality genomic DNA from submitted samples and assess purity/integrity (A260/280, gel or fragment analyzer).
- DNA fragmentation: Randomly fragment DNA to ~100–300 bp by sonication or enzymatic digestion and confirm fragment size distribution.
- 5caC DNA immunoprecipitation (IP): Denature fragmented DNA, incubate with a 5caC-specific antibody, capture immune complexes, and wash under stringent conditions.
- Input background control: Reserve an aliquot of fragmented DNA as input and process it in parallel from library preparation onward to model background.
- Elution, amplification, and library construction: Elute 5caC-enriched DNA, perform limited-cycle PCR, and prepare indexed libraries for both IP and input fractions.
- High-throughput sequencing: Sequence on an Illumina platform at a depth appropriate for genome size and study design.
- Primary data processing: Demultiplex, perform quality filtering and adapter trimming, align reads to the reference genome, and remove duplicates.
IP vs input controls and background reduction
Matched input controls enable:
- Statistical identification of true 5caC-enriched regions above local background
- Assessment of antibody specificity and assay noise
- More accurate cross-sample comparisons, especially when using biological replicates
Together, the wet-lab workflow and computational normalization provide robust and reproducible 5caC DIP-Seq results.
5caC DIP-Seq Data Analysis and Bioinformatic Packages: From Peaks to Pathways
Our 5caC DIP-Seq data analysis is designed to take you from raw reads to pathway-level insight, without requiring your team to manage complex pipelines. We call 5caC peaks relative to input, annotate them to genes and genomic features, compare 5caC profiles across conditions, and then layer on functional analyses such as GO/KEGG enrichment and motif discovery.
The table below summarizes which analysis modules are included in our standard and advanced 5caC DIP-Seq packages.
| Analysis module | Standard package | Advanced package |
|---|---|---|
| Raw data QC and read trimming | ✔️ | ✔️ |
| Read alignment and duplicate removal | ✔️ | ✔️ |
| 5caC peak calling vs input control | ✔️ | ✔️ |
| Peak annotation to genes and features | ✔️ | ✔️ |
| Genomic feature distribution summaries | ✔️ | ✔️ |
| Chromosomal density plots | ✔️ | ✔️ |
| Differential 5caC peak analysis | — | ✔️ |
| GO and KEGG enrichment of associated genes | — | ✔️ |
| Motif discovery in (differential) peaks | — | ✔️ |
| Promoter-centered metagene profiles | — | ✔️ |
| Integration with RNA-seq or ChIP-seq (provided data) | — | Optional add-on |
| Custom figure panels (publication-style) | — | Optional add-on |
Deliverables and Demo Results: What You Receive from a 5caC DIP-Seq Project
All deliverables are provided as structured folders containing raw, processed, and analysis outputs.
- Data files
- FASTQ (on request)
- Aligned BAM (IP and input)
- QC summaries
- Peak and annotation files
- 5caC peak BED files
- Differential peak tables (if applicable)
- Gene/feature annotation tables
- Analysis and visualization
- GO/KEGG and motif reports
- Genomic distribution summaries
- BigWig tracks, metagene plots, publication-ready figures
Genomic distribution of modification peaks.
Chromosomal distribution of modification peak density
GO and KEGG analysis of differentially modified genes
Visualization of modification peaks
Sample Requirements for 5caC DIP-Seq: Accepted Materials, Input Amounts, and Shipping
We accept a variety of input materials. For best results, submit fresh or properly preserved samples with minimal degradation.
Sample Requirement Table
| Sample type | Recommended input | Notes |
|---|---|---|
| Cultured cells | ≥ 2 × 10⁷ cells | Harvest, wash, and snap-freeze pellets in liquid nitrogen. |
| Tissue | ≥ 400 mg | Fresh or snap-frozen tissue blocks; avoid repeated freeze–thaw. |
| Genomic DNA (gDNA) | ≥ 10 µg, OD 260/280 ~1.8–2.0 | High molecular weight DNA in low-salt buffer; no EDTA-rich eluates. |
| IP-enriched DNA | ≥ 4 ng | Pre-enriched fraction from prior IP; provide protocol for context. |
| Whole blood (EDTA) | ≥ 20 mL | Use EDTA tubes; heparin is not recommended for downstream extraction. |
| Other body fluids (e.g., CSF, plasma) | Contact us | We will advise on volumes and DNA extraction strategy. |
Storage and shipping guidelines
- Cells / tissues: Snap-freeze in liquid nitrogen and store at −80 °C.
- DNA: Store at −20 °C for short term; avoid repeated freeze–thaw cycles.
- Shipping: Use sealed 1.5 mL tubes or cryovials with clear labels. Ship DNA on ice packs; ship cells/tissues on dry ice where possible.
5caC vs 5hmC and 5fC DIP-Seq: Choosing the Right Oxidized Cytosine Assay
TET enzymes oxidize 5mC stepwise to 5hmC, 5fC, and 5caC. Each DIP-Seq assay captures a different layer of this demethylation pathway.
Table. Comparison of oxidized cytosine DIP-Seq assays
| Assay | Oxidized base profiled | Dynamics & stability | Typical genomic enrichment | Typical use case |
|---|---|---|---|---|
| 5hmC DIP-Seq | 5-hydroxymethylcytosine (5hmC) | Relatively stable oxidation state | Active enhancers and gene bodies | Map broad hydroxymethylation landscapes and transcription-associated marks |
| 5fC DIP-Seq | 5-formylcytosine (5fC) | More transient than 5hmC; intermediate in demethylation | Regulatory regions and sites of active repair | Study intermediate steps of TET/TDG activity and links to transcription/DNA repair |
| 5caC DIP-Seq | 5-carboxylcytosine (5caC) | Very short-lived; accumulates when TDG or repair is limiting | Distal regulatory elements and poised/bivalent promoters | Pinpoint regions of active demethylation where oxidation outpaces repair |
By combining these assays, you can distinguish regions undergoing gradual oxidation from those where base-excision repair is rate-limiting.
When 5caC DIP-Seq is preferred
Select 5caC DIP-Seq when:
- You want to pinpoint sites of active demethylation, especially under TET or TDG perturbations.
- You are studying regulatory elements (enhancers, bivalent promoters) whose activation may depend on rapid methylation turnover.
- You need a complementary layer to 5hmC DIP-Seq, 5fC DIP-Seq, WGBS, or RNA-seq to mechanistically link methylation dynamics with transcriptional changes.
Case Study
5caC DIP-Seq FAQ: Common Questions on Samples, Data, and Study Design
References
- Shen L, Wu H, Diep D, et al. Genome-wide analysis reveals TET- and TDG-mediated 5-methylcytosine oxidation dynamics. Epigenetics & Chromatin 2013.
- Kisil O, Sergeev A, Bacheva A, Zvereva M. Methods for detection and mapping of methylated and hydroxymethylated cytosine in DNA. Biomolecules 2024.
- Lu X, et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc 2013.