GRO Seq Services | CD Genomics Experts in Transcription Analysis

What Is GRO-Seq?

GRO-Seq (Global Run-On Sequencing) is a powerful next-generation technique that captures the exact position and activity of transcriptionally engaged RNA polymerases by sequencing nascent RNA. Unlike RNA-seq, which only detects mature, steady-state RNA, GRO-seq reveals where transcription is actively occurring—offering a real-time, genome-wide view of gene regulation.

At its core, GRO-seq measures RNA synthesis directly. By pausing transcription in isolated nuclei and restarting it in the presence of labeled nucleotides such as Br-UTP, GRO-seq selectively tags only newly synthesized RNA molecules. These nascent transcripts—often unstable, short-lived, and rapidly degraded in vivo—provide valuable insight into transcription initiation, pausing, elongation, and enhancer activity that cannot be captured by conventional methods.

GRO-Seq vs PRO-Seq – Detailed Comparison

Feature / Scenario	GRO-Seq	PRO-Seq
Nucleotide Labeling	Br-UTP (bromouridine triphosphate)	Biotin-NTP (biotinylated nucleotide triphosphate)
RNA Purification	Anti-BrdU antibody enrichment	Streptavidin bead capture
Resolution	High	Very high (single‐nucleotide precision)
Signal-to-Noise Ratio	Moderate–high	Higher, due to direct capture of paused Pol II
Library Prep Complexity	Moderate	Higher
Primary Focus	Enhancer activity, dynamic transcription initiation	RNA Polymerase II pausing, elongation precision
Common Applications	eRNA detection, transcription initiation profiling	Pol II stalling analysis, stress response transcription monitoring
Choosing This Method If…	You need enhancer usage profiling and bidirectional non-coding RNA detection You want global transcription mapping (promoters, intragenic regions) You prefer a scalable, streamlined protocol for multiple samples/time points	You require single-nucleotide resolution at paused Pol II sites You are investigating transcription under specific stress/controlled stimuli (e.g., heat shock, oxidative stress) You need high specificity for Pol II–centric regulatory events

Not Sure Which One Fits?

Our technical team can help evaluate your experimental goals and recommend the best-fit method. In some cases, a combined GRO-Seq + PRO-Seq strategy may offer complementary insights—balancing broad coverage with nucleotide-level precision.

Sequencing Specifications

Illumina NextSeq CN500
Platform
SE 75
Read Mode
40 M reads/sample
Data Output

Learn more about a wide range of efficient sequencing platforms.

Our GRO-Seq Workflow

Sample Preparation

Nuclei Isolation

From live cells or frozen samples (human, mouse, rat)

Library Preparation

Run-On Transcription

Br-UTP incorporation to label nascent RNA

Sequencing

RNA Enrichment

Magnetic bead-based purification of BrU-labeled transcripts

Data Analysis

Library Preparation

Adapter ligation, reverse transcription, and PCR amplification

Sample Preparation

Sequencing

Illumina NextSeq CN500, SE75, ≥40 million reads/sample

Bioinformatics Analysis

Standard Analysis
Quality control	Adapter trimming, low-quality read filtering, contamination removal
Genome alignment	Strand-specific mapping to the reference genome
Read distribution analysis	Transcript coverage across gene bodies, intergenic, and enhancer regions
TSS-centered meta-analysis	Aggregate signal profiling around transcription start sites
eRNA identification	Detection of bidirectional enhancer RNA

Advanced Analysis
Transcription pausing index	Quantitative assessment of Pol II stalling near promoters
Differential nascent RNA expression	Comparative analysis across experimental conditions
Functional enrichment	GO and KEGG pathway analysis of differentially transcribed genes
Custom analysis (optional)	Motif analysis, elongation rate estimation, or user-defined pipelines

Category	Requirements
Accepted Species	Human, Mouse, Rat
Cell Input	≥ 1 × 10⁷ viable cells per sample Viability ≥ 85%, minimal clumps/debris
Submission Options	Option A: Frozen nuclei (liquid nitrogen, ship on dry ice) Option B: Live cells with culture medium (ambient temperature)
Optional Services	Nuclei isolation service (available on request) Custom sample types accepted upon review

Deliverables & Demo

Demo Figure: Functional Enrichment Overview

Demo Figure: Pol II Behavior & Regulatory Feature

Download Our Demo Report

Raw reads (FASTQ) and QC reports

Genome-aligned files (BAM)

Nascent RNA expression matrix

Differential expression results

Volcano plot, heatmap, scatter plot

GO and KEGG enrichment analyses

RNA Pol II TSS metagene and pause index profiles

eRNA identification and enhancer activity maps

Frequently Asked Questions (FAQ)

- What makes GRO-Seq different from RNA-Seq?
  - GRO-Seq captures nascent (newly synthesized) RNA by labeling paused RNA polymerases, offering a real-time snapshot of transcription activity. In contrast, RNA-Seq profiles mature, steady-state transcripts reflecting cumulative RNA levels and processing.
- Can GRO-Seq detect enhancer RNAs (eRNAs)?
  - Yes. GRO-Seq effectively captures short-lived, bidirectional eRNAs, which are unstable and often missed by RNA-Seq. This enables direct quantification and mapping of active enhancers.
- How does GRO-Seq compare to PRO-Seq?
  - Both methods map nascent transcription, but they differ in labeling chemistry and resolution. GRO-Seq uses Br-UTP, making it ideal for broad transcription profiling, especially enhancers. PRO-Seq uses biotin-NTP, offering higher resolution at precise polymerase pause sites .
- What sample types are supported for GRO-Seq?
  - We accept:
    - Live cells (human, mouse, rat), shipped with culture medium
    - Isolated nuclei, flash-frozen and shipped on dry ice
      Optional nuclei isolation service available—contact our team for support.
- What bioinformatics analyses are included?
  - Our standard pipeline includes:
    - Quality trimming and filtering
    - Strand-specific genome alignment
    - Transcript distribution overview and TSS profiling
    - eRNA detection
    Advanced options include pausing index, differential nascent RNA analysis, pathway enrichment (GO/KEGG), motif discovery, and elongation rate analysis.
- Do you offer custom analyses?
  - Absolutely. We tailor workflows to meet your needs—whether that's novel motif scanning, enhancer-gene network construction, or custom statistical modeling. Just let our team know your goals.
- How do I decide between GRO-Seq and PRO-Seq?
  - - Choose GRO-Seq if you need broad transcriptional profiling, especially enhancer activity (eRNA).
    - Opt for PRO-Seq when you require single-nucleotide resolution at paused RNA polymerase II sites.

Still unsure? Our scientists are happy to advise based on your project design and objectives.

Industry Case Study: GRO-seq Maps Enhancer-Mediated Gene Regulation in Mouse Liver

BMC Genomics (2018), "Nascent RNA sequencing analysis provides insights into enhancer-mediated gene regulation"

DOI: https://doi.org/10.1186/s12864-018-5016-z

Background & Rationale

Understanding how enhancers control gene expression in vivo remains a key challenge in epigenetics. Traditional methods infer enhancer activity via histone modifications or chromatin accessibility, yet enhancer RNAs (eRNAs)—rapidly transcribed, bidirectional RNAs—provide direct evidence of active enhancers. This study applied GRO-seq to wild-type and HDAC3 knockout mouse livers to identify active enhancer elements and how HDAC3 influences enhancer-driven gene regulation.

Core Analytical Dimensions and Technical Findings

Nascent Transcript Profiling: GRO-seq captured genome-wide transcription of both coding genes and eRNAs.
Enhancer Activity Quantification: Identified thousands of eRNAs showing increased transcription upon HDAC3 deletion.
Integrated Multi-omics: Combining GRO-seq with ChIP-seq enabled linking enhancer activity, transcription factor binding, and target gene expression.
Functional Annotation: The study performed a functional enrichment analysis, revealing eRNAs linked to lipid metabolism and liver-specific gene networks.

Summary of features distinguishing NRSA from exiting GRO/PRO-seq analysis tools.

Interpretation and Industry Relevance

This research demonstrates GRO-seq's unique strength for directly measuring enhancer activity in complex tissues under physiologically relevant conditions. Unlike histone-mark-based inference, GRO-seq reports functional output—transcription. By pairing with ChIP-seq and gene expression data, the study provides a comprehensive view of enhancer-mediated gene regulation, informing potential drug targets and therapeutic biomarkers related to liver physiology.

Strategic Takeaway

This BMC Genomics study highlights how GRO-seq:

Enables direct detection of eRNAs, bypassing indirect epigenetic inference.
Supports functional multi-omic integration, crucial for mechanistic insight.
Is suitable for in vivo analyses, not just cell culture.

For researchers exploring enhancer dynamics—whether in disease, metabolism, or development—CD Genomics' GRO-seq service offers the precise resolution and analytical rigor required for advanced functional genomics and regulatory element validation.

GRO-Seq Analysis Services to Profile Nascent RNA Expression

What Is GRO-Seq?

Why GRO-Seq?

When to Use GRO-Seq:

Benefits Over Other Transcriptomic Techniques:

GRO-Seq vs PRO-Seq – Detailed Comparison

Our Capabilities

Sequencing Specifications

Our GRO-Seq Workflow

Nuclei Isolation

Run-On Transcription

RNA Enrichment

Library Preparation

Sequencing

Bioinformatics Analysis

Sample Requirements

Deliverables & Demo

Frequently Asked Questions (FAQ)

Industry Case Study: GRO-seq Maps Enhancer-Mediated Gene Regulation in Mouse Liver

Background & Rationale

Core Analytical Dimensions and Technical Findings

Interpretation and Industry Relevance

Strategic Takeaway