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CUT&Tag-IT® R-loop Assay Kit

Genome-wide profiling of DNA-RNA hybrids in cells

 

CUT&Tag-IT® R-loop Assay Kit Overview

CUT&Tag-IT<sup>®</sup> R-loop Assay Kit
 

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CUT&Tag (Cleavage Under Targets & Tagmentation) is a genome-wide chromatin profiling technique that utilizes an antibody-based enzyme tethering strategy to target specific histone modifications or proteins to reveal chromatin-binding locations for those proteins of interest. Following antibody binding, NGS libraries are prepared in a single step by tagmentation using the protein A-Tn5 (pA-Tn5) transposome enzyme that has been pre-loaded with sequencing adapters.

Active Motif’s CUT&Tag-IT® R-loop Assay Kit utilizes a CUT&Tag-based approach to profile R-loops genome-wide using a specifically optimized protocol for cell samples and our DNA-RNA hybrid (Clone S9.6) antibody. This is not simply the same as our CUT&Tag-IT Assay Kit with a DNA-RNA Hybrid mAb (Clone S9.6). The CUT&Tag-IT R-loop Assay Kit includes optimized DNA Binding Buffer, modified DNA Purification Columns, Q5 Polymerase Master Mix and a second mixing step during secondary antibody incubation all performed in an efficient strip-tube format protocol specifically designed for R-loop targets.

R-loops are three-stranded nucleic acid structures that are formed when a single stranded RNA invades the DNA double helix to hybridize with a complementary DNA strand, leaving the non-template DNA strand unpaired. These structures form normally during transcription initiation, IgG class switching, and mitochondrial DNA replication. They also appear normally at telomeres. Abnormal R-Loop formation is associated with DNA damage, replication fork stalling, impaired DNA repair, and genomic instability. Cancer cells with aberrantly activated transcriptional programs can have transcription-replication conflicts, enhancing the likelihood of abnormal R-loop formation.

CUT&Tag-IT R-loop Assay Kit Advantages:

  • Low background and high signal-to-noise ratio compared with other chromatin profiling techniques
  • Faster and more efficient than DNA:RNA immunoprecipitation (DRIP) methods and chromatin immunoprecipitation (ChIP) methods
  • Compatible with 37,250 to 500,000 fresh or cryopreserved cells

This CUT&Tag-based approach to studying R-loops provides a powerful and highly sensitive new tool for investigating the formation, regulation, and function of R-loops in various biological contexts, ultimately advancing our understanding of this important molecular process.

Learn more about the CUT&Tag Assay and how it works

How the CUT&Tag-IT R-loop Assay Works

How the CUT&Tag-IT R-loop Assay Works

 

CUT&Tag-IT® R-loop Assay Kit Contents

The CUT&Tag-IT® R-loop Assay Kit components are shipped at two temperatures, with one box on dry ice for components to be stored at -20°C, and a second box at room temperature for components that are not to be frozen and must be stored at 4°C. Please store components according to the storage conditions in the manual after first use. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.

Reagents included:

  • 5% Digitonin, store at -20°C
  • R-loop RNase A, store at -20°C
  • Protease Inhibitor Cocktail, store at -20°C
  • CUT&Tag-IT® Assembled pA-Tn5 Transposomes, store at -20°C
  • 10 µg/µL Proteinase K, store at -20°C
  • DNA-RNA Hybrid mAb (Clone S9.6), 1 mg/mL, store at -20°C
  • Rabbit Anti-Mouse Antibody, store at -20°C
  • Tagmentation Buffer, store at -20°C or 4°C
  • Q5 Polymerase Master Mix, store at -20°C
  • i7 Indexed Primer 1, store at -20°C
  • i7 Indexed Primer 2, store at -20°C
  • i7 Indexed Primer 3, store at -20°C
  • i7 Indexed Primer 4, store at -20°C
  • i5 Indexed Primer 1, store at -20°C
  • i5 Indexed Primer 2, store at -20°C
  • i5 Indexed Primer 3, store at -20°C
  • i5 Indexed Primer 4, store at -20°C
  • 1X Binding Buffer, store at 4°C
  • Dig-Wash Buffer, store at 4°C
  • Concanavalin A Beads, store at 4°C
  • SPRI Beads, store at 4°C
  • Dig-300 Buffer, store at 4°C
  • Antibody Buffer, store at 4°C
  • 1% IGEPAL, store at RT
  • 0.5 M EDTA, store at RT
  • 10% SDS, store at RT
  • DNA Purification Wash Buffer, store at RT
  • DNA Purification Elution Buffer, store at RT
  • DNA Purification Binding Buffer, store at RT
  • 3 M Sodium Acetate, store at RT
  • DNA Purification Columns SF, store at RT
 

CUT&Tag-IT® R-loop Assay Kit Data

CUT&Tag-IT R-loop Assay Kit Works with Various Cell Lines
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Figure 1. CUT&Tag-IT R-loop Assay Kit Works with Various Cell Lines
500,000 HEK293, HCT116, SH-SY5Y, T47D, Jurkat, and MCF7 cells were assayed (top six IGV genome browser tracks) in CUT&Tag-IT R-loop Assay Kit showing R-loops at active promoter sites. For comparison, the bottom two browser tracks are 500,000 K562 cells assayed in CUT&Tag-IT Assay Kit Anti-Rabbit (Cat. No. 53160) with anti-H3K27me3 (Cat. No. 39155) and anti-H3K27ac (Cat. No. 39133).

CUT&Tag-IT R-loop Assay Kit specificity is demonstrated by signal depletion after RNAse treatment
(Click image to enlarge)

Figure 2. CUT&Tag-IT R-loop Assay Kit specificity is demonstrated by signal depletion after RNAse treatment
500,000 K562 or HEK293 cells were assayed using the CUT&Tag-IT R-loop Assay Kit in duplicate with or without RNase A treatment. RNase A is expected to digest away R-loops thus reducing signal. The RNase A treatment clearly results in a dramatic decrease in signal, demonstrating that the CUT&Tag-IT R-loop Assay Kit specifically detects R-loops.

CUT&Tag-IT R-loop Assay Kit Specifically Detects R-loops RNase A Treatment
(Click image to enlarge)

Figure 3. CUT&Tag-IT R-loop Assay Kit Specifically Detects R-loops: RNase A Treatment Reduces R-loop Signal but Does Not Impact CTCF CUT&Tag Signal
500,000 HEK293 cells were assayed in duplicate with CUT&Tag-IT R-loop Assay Kit or the CUT&Tag-IT Assay Kit Cat. No. 53160 using anti-CTCF (Cat. No. 61311) with or without RNase A treatment. 500,000 HEK293 cells were also assayed with the CUT&Tag-IT Assay Kit, Anti-Rabbit (Cat. No. 53160) for H3K27me3 (Cat. No. 39155) for comparison (bottom track). The RNase A treatment only decreased signal in the cells assayed with the DNA-RNA Hybrid mAb (Clone S9.6) and did not have any effect on the CTCF signal, showing that the R-loop signal is specific.

 

CUT&Tag-IT® R-loop Assay Kit FAQs

1. Kit Overview & Applications

The CUT&Tag-IT R-loop Assay Kit is specifically optimized for DNA–RNA hybrid (R-loop) profiling, while the standard CUT&Tag-IT Assay Kit is designed for general chromatin targets such as histone modifications.

  • Target specificity: R-loop kit detects DNA–RNA hybrids using S9.6 antibody
  • Optimized reagents: Buffers and purification steps preserve R-loop structures
  • Workflow differences: Enhanced protocol for R-loop sensitivity and stability
Protocol Steps CUT&Tag-IT R-loop Assay Kit CUT&Tag-IT Assay Kit
Input amount 200,000 to 500,000 5,000 to 500,000
Input material Cells Cells
Assay format Strip tubes 1.5 ml tubes
Bind Secondary Antibody 2 x 1 hour 1 hour
Post binding wash steps 3 washes (200 µL each) 3 washes (1 mL each)
Post tagmentation clearnup Modified columns Standard columns
PCR Q5 Polymerase Master Mix Q5 Polymerase
Total cycle number 12 cycles 12-14 cycles

Yes. The kit can be used for other targets using mouse primary antibodies.

  • Compatible with histone modifications
  • Requires mouse primary antibodies
  • Not recommended for transcription factors

2. Sample Preparation & Workflow Setup

Yes. A visible nuclei pellet is not always observed, and you should proceed with the protocol.

  • Pellets may be small or translucent
  • Orient tube caps consistently during centrifugation
  • Handle carefully to avoid disturbing the pellet location

Concanavalin A (Con A) beads should appear evenly suspended and uniformly opaque. When cells bind, the beads become visibly larger, darker, and form soft aggregates that move together in the tube.

  • Unbound beads: fine, evenly dispersed particles
  • Cell-bound beads: larger clusters that settle more quickly
  • Clumping or debris: indicates poor cell prep or bead handling

Watch the short video below for a visual reference of proper bead appearance and binding:

3. Controls & Experimental Design

No. IgG controls are not recommended due to variability in tagmentation-based assays.

  • IgG controls can produce inconsistent results
  • Recommended control: no S9.6 antibody condition

Yes. RNase A-treated samples will still produce a library, but at significantly reduced yield.

RNase A provides efficient signal reduction without the extended incubation required for RNase H.

  • RNase H did not fully eliminate CUT&Tag signal
  • RNase H requires overnight digestion
  • No significant benefit observed vs RNase A

Reference: Wang et al. (2021), Sci. Adv.

4. Sequencing & Data Expectations

Approximately 25 million reads per sample are recommended for robust R-loop profiling.

Mitochondrial reads typically range from ~36% to 67%, depending on cell type and treatment.

5. Data Quality, Troubleshooting & Validation

High background is often caused by poor cell quality or compromised nuclei integrity.

  • Use viable, healthy cells
  • Confirm integrity via Trypan Blue or automated counters
  • Avoid over-handling or harsh lysis conditions

The kit shows strong agreement with published R-loop datasets, with a Pearson correlation of ~0.84.

6. Library Quality & Expected Results

S9.6 samples show characteristic fragment distributions consistent with successful R-loop enrichment.

RNase A-treated samples show reduced signal intensity, confirming depletion of DNA–RNA hybrids.


 

CUT&Tag-IT® R-loop Assay Kit Documents

 

 

You might also be interested in:

US Pat. No. 10,689,643, EP Pat. No. 2999784 and related patents and applications

Permitted Use; Resale Prohibited

In the absence of an express written agreement to the contrary, all products are sold and services deliverables are provided by Active Motif for (a) internal in vitro research purposes only and may not be used for services or any other commercial purpose (b) the exclusive use of the original purchaser, and are not to be resold. You agree not to reverse engineer or otherwise attempt to discover the structure or composition of products or services deliverables unless we otherwise agree in writing.

 
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CUT&Tag-IT® R-loop Assay Kit 16 rxns 53167 ¥20,090 Add to Cart
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CUT&Tag-IT® R-loop Assay Kit Manual