What is the turnaround time for the rPRO-seq service?

While the library preparation workflow itself takes ~15 hours, our standard service turnaround time (including QC, Sequencing, and Bioinformatic Analysis) is typically 4-5 weeks, depending on project scale.

Can rPRO-seq differentiate between primary and secondary drug effects?

Yes. By capturing nascent RNA, rPRO-seq reflects the immediate transcriptional changes caused by a drug (often within 15-30 minutes), whereas standard RNA-seq shows a mix of direct targets and downstream secondary effects.

Is rPRO-seq suitable for analyzing enhancer RNAs (eRNAs)?

Absolutely. eRNAs are often short-lived and non-polyadenylated, making them hard to catch with polyA-RNA-seq. rPRO-seq detects them at the moment of synthesis, providing a robust readout of enhancer activity.

How does the DBO blocking technology work?

We use adapters with a specific 3' modification (DBO) that prevents the ligase enzyme from joining two adapters together. This forces adapters to ligate only to the RNA insert, virtually eliminating the "adapter dimer" peak.

Do I need to perform the nuclear run-on step myself?

No. You simply send us the cell pellets or frozen nuclei. We handle the entire process, including nuclei isolation, nuclear run-on (NRO), library preparation, and sequencing.

rPRO-seq Service: Rapid & Low-Input Nascent RNA Profiling

Capture real-time transcriptional dynamics at single-base resolution from as few as 10^5 cells —powered by proprietary App-DNA & DBO blocking technology.

Unlike standard RNA-seq that only measures steady-state RNA, rPRO-seq captures nascent RNA directly from chromatin, eliminating adapter dimers and delivering high-purity data for Pol II pausing and enhancer activity analysis in just ~15 hours.

Key Highlights:

Ultra-Low Input: Robust data from 10^5 cells (vs. 10^7 for standard PRO-seq).
No Dimers: Proprietary DBO blocking strategy eliminates background noise.
High Speed: Workflow compressed to ~15 hours to minimize RNA degradation.
High Resolution: Single-nucleotide resolution for precise Pol II mapping.

Download Sample Report

rPRO-seq service illustration showing nascent RNA profiling and Pol II dynamics

Overview: From "Snapshot" to "Real-Time Movie"

Transcription is the first and most critical gatekeeper of gene expression. However, standard RNA-seq methods have a fundamental limitation: they measure "steady-state" RNA—the total inventory accumulated in the cell.

Standard RNA-seq is like taking a static photo of a parking lot; you can see the cars, but not their movement. rPRO-seq (Nascent RNA Profiling) is like a high-speed traffic camera. It visualizes the exact density of RNA Polymerase II (Pol II) molecules engaged in transcription, revealing where they are initiating, where they are pausing, and how fast they are moving.

Why "Nascent" Matters: Many critical biological responses occur at the level of transcription rate minutes or hours before total mRNA levels change. rPRO-seq captures these transient events and unstable molecules like Enhancer RNAs (eRNAs), providing a unique window into non-coding genome activity.

Comparison of Nascent RNA Profiling Methods

Dimension	rPRO-seq (Our Service)	Standard PRO-seq	GRO-seq	SLAM-seq
Core Principle	Run-On + Biotin-NTP + App-DNA + DBO Optimization	Run-On + Biotin-NTP labeling + Streptavidin enrichment	Run-On + BrUTP labeling + Immuno-enrichment	4sU metabolic labeling (T>C conversion)
Core Goal	Single-base transient activity & Pol II positioning	Single-base transient activity & Pol II positioning	Genome-wide transient activity & Pol II distribution	RNA synthesis/decay rates & Half-life
Adapter Dimers	None (Blocked by DBO)	Common (High Background)	Low	Low
Input Cells	Low (~10^5)	High (1-2×10^7)	High (1×10^7)	Low
Resolution	Single Base	Single Base	Gene fragment level	Transcript level
Summary	Fast, Low Input, New Strategy prevents dimers, superior data.	High resolution, low background; but long workflow & high input.	Global snapshot; low resolution, long workflow, high input.	Fast, simple, good for half-life; low resolution, cannot map Pol II.

Technical Advantages

The "Adapter Dimer" Breakthrough

We utilize a DBO (3'-blocking) modification on our proprietary App-DNA adapters. This chemical block physically prevents adapters from self-ligating, resulting in near-zero dimer background.

Ultra-Low Input (10^5 Cells)

Optimized chemistry allows sequencing from as few as 100,000 cells (100-fold less than standard PRO-seq). Ideal for rare clinical samples, FACS-sorted populations, and precious specimens.

Accelerated ~15 Hour Workflow

The entire workflow, from nuclei isolation to library prep, is compressed from 4-5 days to ~15 hours. This speed minimizes RNA degradation and maximizes data consistency.

These technical advantages allow researchers to dissect the precise molecular mechanisms governing gene expression with unprecedented sensitivity and speed.

rPRO-seq Workflow Overview

We have optimized the protocol to minimize handling time and maximize data consistency.

Nuclei Isolation: Cells are lysed to isolate nuclei, arresting transcription instantly (Viability >85% required).
Nuclear Run-On (NRO): Transcription resumes in the presence of biotin-11-NTPs; Pol II incorporates biotinylated bases into nascent RNA.
Enrichment: Biotinylated (nascent) RNA is captured using streptavidin magnetic beads, separating it from steady-state RNA.
Library Prep & Sequencing: App-DNA adapters with DBO blocking are ligated to prevent dimers. PE150 sequencing is performed on NovaSeq platforms.

rPRO-seq workflow diagram showing nuclei isolation, nuclear run-on, and DBO-blocked library preparation

Bioinformatics: The Traveling Matrix

Our bioinformatics pipeline goes beyond simple read counting. We employ the Traveling Matrix (TM) analysis to categorize the dynamic state of every gene based on Pol II density in the Promoter vs. Gene Body.

Quadrant	Signal Pattern	Biological Interpretation
I (Pausing)	Promoter: INCREASE Gene Body: DECREASE	Pol II is recruited but cannot escape into the gene body. Indicates a "Pause Release" defect (e.g., CDK9 inhibition).
II (Inhibition)	Promoter: DECREASE Gene Body: DECREASE	The gene is transcriptionally silenced. Pol II is not being recruited to the DNA.
III (Activation)	Promoter: INCREASE Gene Body: INCREASE	The gene is actively turned on. Both recruitment and elongation are upregulated.
IV (Release)	Promoter: DECREASE Gene Body: INCREASE	Pol II is moving efficiently from the promoter into the gene body. The "traffic jam" at the promoter is clearing.

Analytical Strategy: Mechanism & Drug Discovery

rPRO-seq is a powerful tool for dissecting the precise molecular mechanisms governing gene expression.

Uncovering Transcriptional Regulation

Promoter-Proximal Pausing: rPRO-seq maps the critical pause checkpoint 30–50 nt downstream of the TSS. Calculating the Pausing Index (PI) determines if regulation occurs at recruitment or pause release.

Enhancer-Promoter Looping: Active enhancers transcribed into eRNAs are detected with high sensitivity. Correlating eRNA expression with nearby genes allows construction of functional regulatory networks.

Analytical strategy showing promoter-proximal pausing and enhancer-promoter looping detection

Disease Mechanisms and Drug Discovery

Many tumors are "addicted" to high levels of transcription (e.g., MYC-driven). rPRO-seq is the gold standard for evaluating transcriptional kinase inhibitors (CDK9, CDK12/13) because it detects the immediate arrest of Pol II. Furthermore, it differentiates direct drug targets (changes within minutes) from secondary downstream effects.

Applications

Map Active Pol II Distribution

Visualize the exact density of Pol II molecules to understand initiation, pausing, and elongation rates at single-base resolution.

Detect Enhancer RNAs (eRNAs)

Capture unstable eRNAs and upstream antisense RNAs (uaRNAs) before exosome degradation to map non-coding regulatory elements.

Epigenetic & Chromatin Integration

Combine rPRO-seq with ATAC-seq or ChIP-seq/CUT&Tag to determine cause-and-effect: does chromatin opening precede transcription, or vice versa?

Elucidate Drug Mechanisms

Identify primary drug targets by detecting changes in synthesis rates within minutes, distinguishing them from secondary expression shifts.

Neurodevelopmental Studies

Visualize Pol II processivity along exceptionally long genes (>100kb) to reveal elongation defects underlying neurological conditions.

Sample Requirements

Parameter	Requirement	Notes
Sample Type	Cultured Cells	Suspension/Adherent; Fresh or Cryopreserved.
Sample Quantity	≥ 1 × 10^5 cells	5×10^5 recommended for initial optimization.
Cell Viability	> 85%	Critical. Dead cells degrade nascent RNA.
Species	Human, Mouse, Rat	Other eukaryotes (e.g., Drosophila) upon request.

Experimental Design:

Transport: Send as frozen cell pellets or cryopreserved in freezing media on Dry Ice.
Replicates: Three biological replicates per group recommended.
Controls: Minimum of two groups (e.g., control vs treatment) for differential analysis.

Case Study: Validating Low-Input Profiling in Rare Progenitors

Study: Enhancing transcriptome mapping with rapid PRO-seq profiling of nascent RNA (BioRxiv, 2022). This study utilized rPRO-seq to understand the role of INTS11 in rare Hematopoietic Progenitor Cells (HPCs).

rPRO-seq data showing high fidelity mapping of TSS peaks Figure 1. High-Fidelity Mapping
Starting with only 500,000 FACS-sorted cells, rPRO-seq showed clean, sharp peaks at Transcription Start Sites (TSS) with minimal background noise, identifying 3,525 differentially expressed genes.

Upon Ints11 depletion, genes shifted massively into Quadrant I (Pausing). 50% of downregulated genes showed increased Pol II at the promoter but decreased Pol II in the gene body.

Challenge:

Target cells (HPCs) were extremely rare in the bone marrow of the knockout mouse model. Obtaining the millions of cells required for standard PRO-seq was impossible.

Solution:

The team utilized rPRO-seq starting with only 5×10^5 FACS-sorted cells to profile nascent RNA.

Conclusion:

Data conclusively proved that INTS11 is required for the release of paused Pol II into productive elongation. Without rPRO-seq's low-input capability, this insight would have remained undiscovered.

FAQs – Frequently Asked Questions

- What is the turnaround time for the rPRO-seq service?
  - While the library preparation workflow itself takes ~15 hours, our standard service turnaround time (including QC, Sequencing, and Bioinformatic Analysis) is typically 4-5 weeks, depending on project scale.
- Can rPRO-seq differentiate between primary and secondary drug effects?
  - Yes. By capturing nascent RNA, rPRO-seq reflects the immediate transcriptional changes caused by a drug (often within 15-30 minutes), whereas standard RNA-seq shows a mix of direct targets and downstream secondary effects.
- Is rPRO-seq suitable for analyzing enhancer RNAs (eRNAs)?
  - Absolutely. eRNAs are often short-lived and non-polyadenylated, making them hard to catch with polyA-RNA-seq. rPRO-seq detects them at the moment of synthesis, providing a robust readout of enhancer activity.
- How does the DBO blocking technology work?
  - We use adapters with a specific 3' modification (DBO) that prevents the ligase enzyme from joining two adapters together. This forces adapters to ligate only to the RNA insert, virtually eliminating the "adapter dimer" peak.
- Do I need to perform the nuclear run-on step myself?
  - No. You simply send us the cell pellets or frozen nuclei. We handle the entire process, including nuclei isolation, nuclear run-on (NRO), library preparation, and sequencing.

References:

Cingaram, P. K. R., Beckedorff, F., Yue, J., et al. Enhancing transcriptome mapping with rapid PRO-seq profiling of nascent RNA. bioRxiv, 2022.07.06.498888 (2022).
Ekstrom, T. L., et al. Glucocorticoid receptor suppresses GATA6-mediated RNA polymerase II pause release to modulate classical subtype identity in pancreatic cancer. Gut, 74(7), 1112-1124 (2025).
Dastidar, S. G., et al. Transcriptional responses of cancer cells to heat shock-inducing stimuli involve amplification of robust HSF1 binding. Nature Communications, 14(1), 7420 (2023).
Mahat, D. B., et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using PRECISION nuclear run-on (PRO-seq). Nature Protocols, 11(8), 1455–1476 (2016).