1. What types of samples can I submit for 5caC DIP-Seq?

We accept cultured cells, tissues, whole blood (EDTA), genomic DNA, and pre-enriched IP DNA. See the sample requirement table above for recommended input amounts and storage conditions.

2. How much material do I need for a typical project?

As a guide, we recommend ≥2 × 10⁷ cells, ≥400 mg tissue, or ≥10 µg high-quality gDNA per sample. For IP-enriched DNA, ≥4 ng is sufficient. If your available material is limited, contact us to discuss feasibility and optimization strategies.

3. What data and reports will I receive?

You receive sequencing data (if requested), aligned BAM files, 5caC peak calls, differential peak tables (for multi-group projects), GO/KEGG and motif analysis results (for advanced packages), and visualization files such as BigWigs and summary figures.

4. Can you integrate 5caC DIP-Seq with other datasets?

Yes. We routinely integrate 5caC maps with RNA-seq, ChIP-seq, ATAC-seq, and DNA methylation data to explore how active demethylation relates to transcription, chromatin accessibility, and histone marks.

5. How many biological replicates do you recommend?

For comparative studies, we generally suggest at least two biological replicates per condition to enable robust differential 5caC analysis and statistical interpretation. Additional replicates further increase power and reliability.

6. What is the typical turnaround time?

Turnaround depends on sample number, sequencing depth, and analysis complexity, but full projects are usually completed within a multi-week timeframe from sample acceptance. We will provide an estimated range during project scoping.

7. Is this service suitable for clinical diagnostics?

No. Our 5caC DIP-Seq service is intended for research use only (RUO) and is not validated for clinical diagnostic applications, patient management, or direct therapeutic decision-making.

8. How do I get started or request pricing?

You can start by sharing your organism, sample types, number of conditions, and key biological questions. Our scientific team will propose a tailored 5caC DIP-Seq design, recommend an analysis package, and provide a formal quote.

5caC DIP-Seq Service - CD Genomics

Overview

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.

Schematic showing TET oxidation of 5mC to 5hmC, 5fC, and 5caC, with TDG/BER removing 5fC/5caC back to cytosine 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.

Applications

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.

Principles

Technical Principles: How 5caC DIP-Seq Enriches and Maps 5-Carboxylcytosine

DNA immunoprecipitation with anti-5caC

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.

High-throughput sequencing and peak detection

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.

Comparing 5caC patterns across conditions

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.

Advantages

Why Choose Our 5caC DIP-Seq Service: Sensitivity, QC, and Expert Support

One-stop 5caC DIP-Seq service

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.

Optimized IP workflow and strict QC

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.

Professional bioinformatics support

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.

Workflow

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.

Vertical flowchart showing the 5caC DIP-Seq workflow from DNA extraction and fragmentation through 5caC IP and input control to library construction, sequencing, and data processing.

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.

Data

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

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

Pie chart showing the genomic distribution of modification peaks (promoter, upstream, intron, exon, intergenic). Genomic distribution of modification peaks.

Bar-style chromosome plot showing red fragment-count density of modification peaks across chr1–chr22, chrX, chrY, and chrM. Chromosomal distribution of modification peak density

Bar plot of enriched GO biological processes and bubble plot of KEGG pathways for differentially modified genes. GO and KEGG analysis of differentially modified genes

Genome browser tracks showing input and IP signals for control and SNI conditions, with a highlighted modification peak at the Gtse1 gene. Visualization of modification peaks

Samples

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.

Comparison

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

Case Study

TET/TDG-Dependent 5mC Oxidation Dynamics in Mouse ESCs

Reference: Shen L, Wu H, Diep D, et al. Genome-wide analysis reveals TET- and TDG-mediated 5-methylcytosine oxidation dynamics. Epigenetics & Chromatin. Published 18 March 2013

Background

To understand how extensively active DNA demethylation operates in the mammalian genome, Shen et al. generated genome-wide maps of 5mC, 5hmC, 5fC, and 5caC in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs).

Methods

Genomic DNA from wild-type and TDG-knockout ESCs was fragmented.
Modification-specific antibodies were used to enrich DNA carrying 5mC, 5hmC, 5fC, or 5caC (DIP-style IP).
The enriched fractions were subjected to high-throughput sequencing, and genome-wide enrichment profiles were generated.

Our 5caC DIP-Seq service uses a conceptually similar enrichment-plus-sequencing approach, optimized for sensitivity and QC in service settings.

Results

In wild-type ESCs, 5fC and 5caC signals were primarily detectable at repetitive sequences, with much lower levels at single-copy regions.
In TDG-deficient ESCs, both 5fC and 5caC accumulated strongly at distal regulatory elements and repressed/poised promoters, indicating ongoing TET oxidation and impaired repair at these sites.
Comprehensive mapping of all four cytosine states (5mC, 5hmC, 5fC, 5caC) revealed widespread TET/TDG-dependent cycling across the genome.

Conclusion

This study demonstrates that 5caC DIP-like profiling can sensitively report where TET/TDG-mediated demethylation is active across the ESC genome. By applying our 5caC DIP-Seq service to your models—whether gene-edited cell lines, differentiation systems, or other perturbations—you can uncover similar patterns of active demethylation at regulatory regions of interest.

Accumulation of 5fC and 5caC in Nonrepetitive Regions in Tdg-Deficient Mouse ESCs

FAQ

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.

5caC DIP-Seq Service: Genome-wide Mapping of Active DNA Demethylation