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Tn5-Mediated R-Loop CUT&Tag Service: Ultra-Low Input Profiling

Analyze R-loops in rare and precious samples with our R-loop CUT&Tag Service. Utilizing Tn5 transposase technology, we achieve ultra-low input profiling (down to 1,000 cells) with high resolution and minimal background. The ideal solution for clinical biopsies, stem cells, and detailed regulatory mapping. RUO.

  • Ultra-Low Input: Profiling from as few as 1,000 cells.
  • Tn5 Tagmentation: Simultaneous cleavage and adapter ligation in situ.
  • High Fidelity: Exceptional signal-to-noise ratio with no sonication bias.
  • Flexible Targeting: Compatible with S9.6 antibody or HBD sensors.
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3D illustration of R-loop CUT&Tag mechanism with Tn5 transposase.

Overview: Ultra-Sensitive R-Loop Mapping with Tn5

R-loops are three-stranded structures (DNA-RNA hybrid + single-stranded DNA) that play key roles in gene regulation and genomic stability. However, standard mapping methods like DRIP-seq typically require millions of cells and harsh sonication, making them unsuitable for rare samples or clinical biopsies.

Our Tn5-Mediated R-Loop CUT&Tag Service (Cleavage Under Targets and Tagmentation) revolutionizes this process. Instead of extracting and sonicating DNA, we work inside intact nuclei. We use an antibody (typically S9.6) to guide a pA-Tn5 transposase enzyme directly to the R-loop sites. Once there, the transposase "cuts and tags" the DNA with sequencing adapters in a single step.

This "in situ" chemistry dramatically boosts sensitivity and reduces background noise. It allows us to generate high-resolution R-loop maps from as few as 1,000 cells, preserving the native chromatin environment and revealing regulatory R-loops that other methods miss.

Service Snapshot

  • Target: Native R-loops
  • Chemistry: pA-Tn5 Transposase
  • Input: Ultra-low (1k-50k cells)
  • Key Benefit: High Sensitivity

Why Choose Tn5-Mediated CUT&Tag?

Unmatched Sensitivity

Because the Tn5 transposase amplifies the signal directly at the target site within the nucleus, our service can profile rare populations—such as patient-derived xenografts (PDX), stem cells, or specific immune subsets—that were previously impossible to analyze.

High Fidelity & Low Background

CUT&Tag washes away unbound antibody before the sequencing library is generated. Only the DNA immediately adjacent to the R-loop is tagged and sequenced. This results in data with a very high signal-to-noise ratio.

Preserving Native Structure

Harsh sonication can disrupt fragile chromatin features. Our Tn5-mediated workflow is gentle and performed on permeabilized nuclei under physiological conditions, preserving native R-loop conformation.

Technical Comparison: CUT&Tag vs. MapR vs. DRIP

Feature R-Loop CUT&Tag (This Service) MapR DRIP-seq
Core Chemistry Tn5 Transposase (Tagmentation) MNase (Enzymatic Cleavage) Sonication (Physical Shearing)
Input Sensitivity Ultra-Low (1k–50k cells) Low (10k–100k cells) High (>1M cells / µg DNA)
Background Noise Very Low Low Moderate
Resolution High (Base-pair precision) High (MNase footprint) Low/Medium (Broad peaks)
Workflow In situ (Intact Nuclei) In situ (Intact Nuclei) In vitro (Purified gDNA)
Best For Rare/Precious Samples Rapid screening Global abundance comparisons

Our Workflow: From Nuclei to Libraries

1. Nuclei Preparation

Cells are harvested and gently permeabilized (or nuclei are isolated from tissue) to allow reagents to enter the nucleus without disrupting chromatin.

2. Target Recognition

The specific S9.6 antibody (or a custom hybrid-binding domain) binds to the R-loops in situ. Secondary antibodies are added to amplify the docking site.

3. pA-Tn5 Docking

The Protein A-Tn5 transposase fusion protein is added. Protein A binds tightly to the antibody, positioning the transposase exactly at the R-loop.

4. Tagmentation

Magnesium is added to activate the transposase. It simultaneously cuts the DNA and ligates sequencing adapters ("tagmentation") only at the R-loop sites.

5. Library Amplification

The tagged DNA fragments are released and amplified via PCR. Because adapters are already attached, the library prep is extremely fast and efficient.

Step-by-step workflow of Tn5-mediated R-loop CUT&Tag from nuclei preparation to library amplification.

Sample Requirements

Sample Type Recommended Input Minimum Input Storage/Transport
Cell Lines 50,000 cells 1,000 cells* Cryopreserved (DMSO) or Flash Frozen pellet
Primary Cells (PBMC, Stem Cells) 50,000 cells 5,000 cells* Fresh or Cryopreserved (Vitality >85%)
Frozen Tissue 5-10 mg ~2 mg Flash Frozen (Liquid Nitrogen)
FACS Sorted Cells 50,000 cells 1,000 cells Spun down immediately & frozen

*Note: For ultra-low input (<5,000 cells), we use a specialized library amplification protocol. Please consult our technical team before shipping.

Key Deliverables

High-Resolution Signal Tracks
BigWig files showing sharp peaks at Transcription Start Sites (TSS) with virtually zero background.

Peak Annotations
BED files of significant R-loop sites, annotated by genomic feature (Promoter, Intron, Terminator) and nearest gene.

Correlation Analysis
Plots showing the correlation between R-loop signal and your RNA-seq data (if provided).

Motif Analysis
Identification of DNA sequence motifs (e.g., G-rich sequences) enriched under the peaks.

Case Study: Resolving Promoter-Proximal R-Loops

Standard DRIP-seq often struggles to detect R-loops at gene promoters because these structures can be transient and are easily lost during harsh DNA extraction. A 2021 study sought to map these "regulatory" R-loops with higher precision to understand their role in pausing RNA Polymerase II.

The researchers employed R-loop CUT&Tag using a specific sensor. They performed the assay in intact nuclei to preserve the delicate promoter structures.

The CUT&Tag data revealed thousands of sharp, high-intensity R-loop peaks centered exactly at the Transcription Start Sites (TSS) of active genes. In comparison, standard DRIP-seq data from the same cells showed broad, diffuse signals that often missed the TSS entirely. The high resolution of CUT&Tag allowed the authors to correlate these R-loops directly with RNA Polymerase II pausing indices.

Comparison of CUT&Tag peaks vs DRIP peaks showing superior resolution at promoters.

Tn5-mediated profiling (CUT&Tag) provides a superior, high-resolution map of promoter-associated R-loops, making it the preferred method for studying transcriptional regulation.

(Source: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor, Science Advances, 2021. CC BY 4.0)

Demo Results (Representative Examples)

IGV tracks showing sharp R-loop CUT&Tag peaks at TSS compared to DRIP-seq.CUT&Tag vs DRIP

Frequently Asked Questions

References

  1. Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor. Science Advances. 2021.
  2. Genome-Wide Native R-Loop Profiling by R-Loop Cleavage Under Targets and Tagmentation (R-Loop CUT&Tag). Methods in Molecular Biology. 2022.
  3. Benchmark of chromatin–protein interaction methods in haploid round spermatids. Frontiers in Cell and Developmental Biology. 2025.
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