What is the minimum sample input for PRO-seq?

For standard PRO-seq, we recommend 1 to 5 million cells per replicate to ensure high library complexity; however, for difficult samples like FACS-sorted cells, we offer modified low-input protocols.

Can you handle frozen tissue samples?

Yes, but flash-frozen samples are required. Slow freezing damages the nuclear machinery. We use specific protocols to extract nuclei from frozen tissue while maintaining RNA Polymerase activity.

Does the analysis include enhancer identification?

Yes. Our advanced bioinformatics pipeline includes tools like dREG to identify active enhancers based on their signature divergent transcription pattern.

Can PRO-seq detect non-coding RNA?

Absolutely. PRO-seq captures all transcription by Pol II, including mRNA, lncRNA, and eRNA. It is superior to RNA-seq for non-coding RNAs because it captures transcripts even if they are not poly-adenylated or are unstable.

Precision Run-On Sequencing (PRO-seq) Service

CD Genomics offers a comprehensive Precision Run-On Sequencing (PRO-seq) Service that captures nascent RNA at single-nucleotide resolution.

Unlike standard methods that only count mature RNA, PRO-seq maps the exact location of active RNA Polymerase II (Pol II) on the genome, allowing you to see transcription exactly while it is happening.

Key Highlights:

High Resolution: Map the exact 3' end of the growing RNA with biotin-based precision.
Mechanism Focused: Perfect for studying pause-release dynamics and enhancer activity.
Complete Solution: From cell nuclei isolation to publication-ready dREG analysis.
Research Use Only: This service is for Research Use Only (RUO).

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Precision Run-On Sequencing illustration showing Pol II pausing and nascent RNA mapping

Why PRO-seq Comparison Advantages Workflow Analysis Strategy Applications Case Study FAQs Inquiry

See Transcription Happening in Real-Time

Traditional RNA sequencing (RNA-seq) is like taking a photo of a busy highway after all the cars have parked. It shows you the final result (steady-state mRNA), but it misses the movement.

Precision Run-On Sequencing (PRO-seq) is different. It acts like a high-speed camera that captures transcription exactly while it is happening.

By isolating nuclei and using specific Biotin-NTPs to "freeze" RNA Polymerase II, we provide a map with single-nucleotide resolution. You can see exactly where the Polymerase paused, where it is moving fast, and where it is waiting.

PRO-seq vs. GRO-seq & RNA-seq

Feature	Standard RNA-seq	GRO-seq	PRO-seq
Target	Steady-state mRNA (Mature)	Nascent RNA (Active)	Nascent RNA (Active)
Resolution	Low (Exon level)	Medium (>50 bp)	Single-Nucleotide (1 bp)
Labeling	None (cDNA conversion)	Br-UTP (Run-on)	Biotin-NTP (Chain Terminator)
3' End Mapping	Poor	Poor	Precise
Enhancer Detection	Very Low	Good	Excellent (dREG compatible)
Pausing Analysis	Impossible	Limited	Gold Standard

Technical Advantages

Single-Nucleotide Resolution

We map the exact 3' end of the growing RNA. As soon as a Biotin-NTP is added, the Polymerase halts, ensuring the read end corresponds exactly to the enzyme's active site.

Direct Detection of Active Enhancers

Because enhancer RNAs (eRNAs) degrade in minutes, standard RNA-seq misses them. PRO-seq captures these unstable transcripts, allowing direct identification of regulatory elements.

Immediate Transcriptional Response

See the "first wave" of transcription. If you are studying a fast-acting drug or stress signal, PRO-seq shows changes in gene expression in as little as 10 minutes.

These advantages make PRO-seq the premier tool for studying transcriptional dynamics, pausing indices, and divergent transcription.

PRO-seq Workflow Overview

Our PRO-seq workflow is rigorous and optimized to ensure high-quality data from nuclei to sequencing.

Nuclei Isolation – We gently isolate nuclei using specialized buffers to keep RNA Polymerase bound to DNA.
Nuclear Run-On – Nuclei are incubated with Biotin-11-NTPs. The Polymerase incorporates these tags and halts immediately.
Streptavidin Capture – Magnetic beads separate the biotin-labeled "nascent" RNA from the unlabeled "old" RNA.
Library Preparation – 3' and 5' adapters are ligated to the purified nascent RNA, followed by reverse transcription.
Sequencing & Analysis – High-depth sequencing identifies the precise 3' ends, followed by custom bioinformatics mapping.

PRO-seq workflow diagram showing nuclear run-on with biotin-NTPs and streptavidin capture

Bioinformatics and Data Analysis

Analysis Type	Content Description
Standard Analysis
1. Raw Data QC	FastQ file generation and quality verification.
2. Mapping Statistics	Alignment rates to the reference genome (genome-wide).
3. BigWig File Generation	Signal tracks for visualization in IGV (Red: + strand, Blue: - strand).
Advanced Analysis
1. Pausing Index Calculation	Calculate the ratio of reads at the promoter vs. gene body to identify stalled genes.
2. dREG Analysis	Detect active enhancers genome-wide based on bidirectional transcription signatures.
3. Metagene Plots	Composite plots showing average Pol II distribution across gene sets.
4. Differential Expression	Identify genes upregulated or downregulated at the transcription level (not just steady-state).
5. Antisense Transcription	Quantify divergent transcription events at promoters and enhancers.

Bioinformatics for PRO-seq differs significantly from RNA-seq. Our custom pipeline handles the specific requirements of nascent RNA data to deliver publication-ready figures.

Analytical Strategy: The "Nascent" Advantage

Understanding gene regulation requires distinguishing between RNA stability and actual production. Traditional RNA-seq measures the total inventory, which is slow to change.

The "Freeze-Frame" Concept

How do we capture an enzyme moving at 2,000 bases per minute? The secret is in the chemistry.

Biotin-NTPs as Chain Terminators

Unlike GRO-seq, which uses a generic label allowing the polymerase to run for ~100 bases (creating a "smear"), PRO-seq uses Biotin-NTPs. These act like a stop sign. As soon as one is added, the Polymerase halts.

Precision Mapping

This "freezing" effect means the end of the RNA read corresponds exactly to the active site of the enzyme. This precision allows calculation of the Pausing Index, determining if Polymerase is stuck at the promoter or running freely.

Comparison of GRO-seq smear vs PRO-seq single nucleotide resolution

Analytical Flow

Input – Nuclei from control vs. treated samples.
Processing – Biotin-run-on, fragmentation, and streptavidin enrichment.
Integration – Mapping reads to identify divergent transcription and pause sites.
Output – List of active enhancers, paused genes, and immediate transcriptional changes.

Applications & Mechanisms

Our PRO-seq service is designed for researchers who need to explain how gene expression is controlled, widely used in developmental biology and cancer research.

Map Pol II Pausing & Release

Determine if your gene is regulated by "release factors" like P-TEFb or c-Myc. A high pausing index indicates the Polymerase is recruited but stalled at the promoter.

Identify Active Enhancers (eRNA)

Detect unstable enhancer RNAs (eRNAs) that standard RNA-seq misses. We use dREG to predict which enhancers are regulating specific genes.

Resolve Primary vs. Secondary Effects

In standard RNA-seq, it's hard to tell which gene turned on first. PRO-seq captures the "first wave" of transcription, distinguishing primary targets from downstream effects.

Antisense Transcription Analysis

Capture RNA being made in the "wrong" direction (divergent transcription), a key regulatory feature often invisible in other assays.

Difficult & Low-Input Samples

For samples with limited material (rare cells, sorted populations), we offer optimized rPRO-seq workflows to handle lower input quantities.

Deliverables:

Raw Data (FastQ) and Aligned Data (BAM)
BigWig files for genome browser visualization
Comprehensive project report with QC steps
Advanced Analysis (dREG, Pausing Index) upon request

Sample Requirements

Sample Type	Recommended Amount	Condition
Cell Lines	> 2 million cells	Fresh or Flash Frozen Pellet
Tissue	> 50 mg	Flash Frozen immediately
Nuclei	> 1 million nuclei	Permeabilized & Frozen

Important Notes:

Freezing: Flash-freezing is required. Slow freezing damages the nuclear machinery essential for the run-on reaction.
Replicates: We recommend at least 2 biological replicates per condition, though 3 is preferred for statistical power.
Transport: Ship on dry ice. Do not send live cells unless arranged specifically.

Case Study: Deciphering Transcriptional Pausing

The following case study illustrates the technical capabilities of PRO-seq based on seminal methodology. [cite_start]It serves as a technical benchmark for what clients can expect from high-resolution nascent RNA mapping[cite: 458].

Background of PRO-seq case study showing heat shock gene regulation model Figure 1. Background & Methodology
The study utilized PRO-seq to map RNA Polymerase II positions in human cells before and after heat shock, using Biotin-NTPs to label nascent RNA 3' ends with single-base precision.

PRO-seq data showing Pol II pausing at promoter before heat shock Figure 2. Paused Polymerase State
Under normal conditions, Pol II was found sitting at the promoter region of heat-shock genes (e.g., HSP70). It had started transcription but stalled after about 30-50 bases.

PRO-seq signal shift demonstrating rapid release of Polymerase into gene body Figure 3. Rapid Release Dynamics
Within minutes of heat shock, the PRO-seq signal shifted. The peak at the promoter decreased, and a wave of signal moved into the gene body, showing "escape" from the pause site.

Conclusion graphic showing pause release as the limiting step in gene regulation Figure 4. Conclusion
PRO-seq proved that the limiting step was Pause Release, not recruitment. This level of kinetic detail would have been invisible with standard RNA-seq.

Background

Researchers needed to determine if RNA Polymerase was recruited after stress or if it was already pre-loaded and waiting.

Methodology

Biotin-NTP labeling provided a high-resolution snapshot of active polymerases across the genome.

Results: Pause Release

The data clearly showed the Polymerase "escaping" the pause site, confirming that release factors are the key regulators.

Significance

This study established PRO-seq as the premier tool for deciphering immediate transcriptional responses and regulatory kinetics.

FAQs – Frequently Asked Questions

- What is the minimum sample input for PRO-seq?
  - For standard PRO-seq, we recommend 1 to 5 million cells per replicate to ensure high library complexity; however, for difficult samples like FACS-sorted cells, we offer modified low-input protocols. Please consult our team for details.
- Can you handle frozen tissue samples?
  - Yes, but flash-frozen samples are required. Slow freezing damages the nuclear machinery. We use specific protocols to extract nuclei from frozen tissue while maintaining RNA Polymerase activity.
- How many biological replicates do I need?
  - We recommend at least 2 biological replicates per condition, though 3 is better for statistical power. Because PRO-seq detects direct transcription with less noise than RNA-seq, 2 replicates are often sufficient for robust differential analysis.
- Does the analysis include enhancer identification?
  - Yes. Our advanced bioinformatics pipeline includes tools like dREG (Detection of Regulatory Elements using GRO-seq/PRO-seq data) to identify active enhancers based on their signature "divergent transcription" pattern.
- Can PRO-seq detect non-coding RNA?
  - Absolutely. PRO-seq captures all transcription by Pol II, including mRNA, lncRNA, and eRNA. It is superior to RNA-seq for non-coding RNAs because it captures transcripts even if they are not poly-adenylated or are unstable.
- Can PRO-seq differentiate between sense and antisense transcription?
  - Yes. PRO-seq is a strand-specific method. Library preparation preserves directionality, allowing you to see reads mapping to forward and reverse strands separately, which is crucial for identifying divergent transcription.
- Do you provide the raw data?
  - Yes, you own the data. We provide raw FastQ files, aligned BAM files, and processed BigWig files for visualization, along with a full project report detailing QC steps and analysis results.

References:

Mahat, D. B., et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nature Protocols (2016).
Kwak, H., et al. Precise maps of RNA polymerase reveal how promoters control initiation and pausing. Science (2013).
Core, L. J., et al. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science (2008).

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