How much starting material is required for NET-Seq?

Typically, we recommend at least 10 million (10^7) cells per sample. This amount is necessary because the Immunoprecipitation (IP) step recovers only the tiny fraction of RNA that is actively being transcribed. However, we have optimized protocols for lower inputs (down to 1-5 million cells) for specific sensitive cell lines.

What is the difference between NET-Seq and PRO-Seq?

The main difference is the "Run-On" step. PRO-seq isolates nuclei and "restarts" transcription in a tube using biotin-labeled nucleotides. NET-Seq skips this artificial step and sequences the RNA exactly as it was inside the cell (Native).

Can you handle immunoprecipitation of specific Pol II modifications?

Yes. Our service allows for the use of specific antibodies. While "Total Pol II" is standard, we can also perform mNET-seq using antibodies against Ser5P or Ser2P phosphorylation states.

Does the service include bioinformatics for pausing indices?

Yes. Our standard deliverable includes a comprehensive analysis package: raw data (FASTQ), aligned reads (BAM), and a detailed report containing Pausing Indices, Metagene Plots, and gene body coverage heatmaps.

NET-Seq Service: Native Elongating Transcript Sequencing

Unlock the dynamics of transcription with our NET-Seq service, offering single-nucleotide resolution of Native Elongating Transcripts without artificial run-on assays. We provide optimized library preparation for mammalian and yeast samples.

This specialized technique captures RNA exactly as it is being synthesized by RNA Polymerase II (Pol II), enabling researchers to pinpoint Pol II positions with single-nucleotide precision.

Key Highlights:

Native Resolution: Map Pol II position exactly at the 3' end of nascent RNA.
High Specificity: Proprietary immunoprecipitation minimizes rRNA contamination.
Advanced Analysis: Pipelines for pausing index and splicing intermediate visualization.
Flexible Input: Optimized protocols for limited cell numbers.

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Diagram showing NET-Seq capturing nascent RNA from Pol II versus standard RNA-seq.

Overview: Capture Nascent RNA at Single-Nucleotide Resolution

Transcriptional regulation is a highly dynamic process, comparable to a high-speed factory assembly line. Standard RNA sequencing methods typically capture the "finished product"—mature, stable mRNA. While valuable, this approach misses the critical "production" details.

Native Elongating Transcript Sequencing (NET-Seq) is a specialized technique designed to capture RNA exactly as it is being synthesized by RNA Polymerase II (Pol II). Unlike other methods that require artificial "run-on" reactions in a test tube, NET-Seq analyzes the native complex directly from living cells.

Our NET-Seq service provides a high-resolution snapshot of the nascent transcriptome. By mapping the exact 3' end of the growing RNA chain, we enable researchers to pinpoint Pol II positions with single-nucleotide precision. This service is ideal for studying transcription elongation rates, Pol II pausing, and co-transcriptional RNA processing.

For researchers interested in alternative metabolic labeling methods for nascent RNA, please see our SLAM-seq service.

Technical Comparison: NET-Seq vs. PRO-Seq

Feature	NET-Seq (Native)	PRO-Seq (Run-On)
Core Principle	Captures Native Pol II complex directly from cells	Uses a Run-On reaction in nuclei
Elongation State	Snapshots the exact position at the moment of lysis	Restarts transcription artificially with Biotin-NTPs
Resolution	Single-nucleotide (3' end mapping)	Single-nucleotide (3' end mapping)
Bias	Minimal (No enzymatic incorporation bias)	Potential bias from Biotin-NTP speed
Manipulation	Less manipulation (Physiological state)	More manipulation (Nuclei isolation)
Best For	Studying native pausing & splicing kinetics	Mapping active polymerase density with high signal

Why Choose Our NET-Seq Service?

Native Resolution without Run-On Bias

We capture the true biological state of transcription. By avoiding in vitro run-on steps, we eliminate the potential for artificial elongation or "run-on" bias that can skew pausing indices.

Superior Library Purity & Specificity

A common failure mode in NET-Seq is "noise" from rRNA. Our proprietary immunoprecipitation (IP) wash buffers are optimized to stringently remove non-specific binding while preserving the fragile RNA-Pol II interaction.

Optimized for Difficult Samples

Standard protocols often require massive sample inputs. We have refined our library preparation chemistry to handle lower input amounts while maintaining high library complexity.

Workflow: From Cell Lysis to Bioinformatic Insights

Our end-to-end service is designed to be simple for the user but rigorous in the lab.

Step 1: Cell Lysis – Cells are lysed under optimized conditions with inhibitors to isolate the chromatin-bound fraction.
Step 2: Immunoprecipitation – We use specific antibodies (e.g., Ser5P, Ser2P) to pull down the Pol II complex without cross-linking.
Step 3: Linker Ligation – A specialized DNA linker is ligated to the 3' end of the nascent RNA, marking the exact position.
Step 4: Library Construction – RNA is eluted, reverse-transcribed, and circularized/ligated for sequencing with minimized PCR duplicates.
Step 5: Sequencing – High-throughput sequencing (PE150) on Illumina NovaSeq to generate deep coverage (>50M reads).
Step 6: Bioinformatics – Raw data processing, PCR duplicate removal, and precise mapping to the genome.

(Note: For researchers needing global run-on sequencing instead of native profiling, please compare this with our GRO-sequencing service.)

NET-Seq workflow from cell lysis to data analysis

Sample Requirements & Submission Guidelines

Parameter	Standard Requirement	Technical Notes
Sample Type	Cultured Cells (Human/Mouse/Yeast)	Suspension or Adherent cells. Tissues require optimization.
Input Amount	> 1 × 10⁷ cells per sample	High input needed for IP of specific protein complexes.
Sample Condition	Fresh Cell Pellets or Flash-Frozen	Harvest quickly on ice to "freeze" transcriptional states.
Purity	Cell Viability > 90%	Dead cells contain degraded RNA which contributes to background noise.
Shipping	Dry Ice	Must remain deep-frozen (-80°C) at all times during transit.

Important: Always label tubes clearly with sample ID, cell count, and date. Download our full Sample Submission Form after requesting a quote.

Case Study: Deciphering Co-transcriptional Splicing Kinetics

Background

Understanding how the spliceosome interacts with RNA Polymerase II is crucial for gene regulation studies. Traditional RNA-seq cannot distinguish whether splicing happens during or after transcription.

Methodology

Using mammalian NET-Seq (mNET-seq), researchers mapped the topology of the Pol II complex associated with phosphorylated C-terminal domains (CTD). As demonstrated in foundational research (Nojima et al., 2015), this method captures RNA intermediates tethered to the polymerase.

Results & Conclusion

The data revealed a distinct accumulation of Pol II reads at the 3' end of exons. This "pausing" behavior suggests that the polymerase waits for the splicing machinery to recognize the splice site before moving forward. NET-Seq successfully visualized the timing of splicing at single-nucleotide resolution.

NET-Seq analysis of co-transcriptional splicing kinetics

Applications of NET-Seq Analysis

NET-Seq provides a "microscope" for the genome, allowing you to visualize regulatory mechanisms that standard Total RNA-seq cannot reveal.

Transcription Elongation & Pausing

Calculate the Pausing Index (PI) to reveal where the polymerase speeds up or slows down, identifying regions of stalled elongation.

Co-transcriptional Splicing Kinetics

Capture transient splicing intermediates and detect "lariat" structures to identify the exact order of intron removal.

Enhancer RNA (eRNA) Detection

Capture unstable RNAs like eRNAs while they are still attached to Pol II, revealing distinct bidirectional transcription signatures.

Transcription Termination

Precisely map the 3' ends of transcripts at termination zones to observe Pol II slowing down downstream of the polyadenylation signal.

Demo Results: High-Fidelity Data Visualization

We believe in data transparency. Below are examples of the high-fidelity visualizations included in our standard analysis package.

Metagene profile illustrating Pol II pausing density at the Transcription Start Site (TSS). Figure 1. Metagene Plots
Visualizing the average Pol II distribution across thousands of genes. A sharp peak at the TSS indicates normal promoter-proximal pausing.

Single-nucleotide mapping of splicing intermediates showing clean exon-intron boundaries. Figure 2. Single-Base Resolution Tracks
IGV-compatible files (.bw) allow you to zoom in to single-nucleotide resolution to verify specific pause sites or splice junctions.

Pausing Indices Calculation

A quantitative table listing the Pausing Index for every expressed gene, allowing you to filter for genes with "stalled" vs. "productive" elongation.

Splicing Efficiency Metrics

Analysis of reads spanning exon-intron junctions to estimate the efficiency of co-transcriptional splicing.

FAQs – Frequently Asked Questions

- How much starting material is required for NET-Seq?
  - Typically, we recommend at least 10 million (10^7) cells per sample. This amount is necessary because the Immunoprecipitation (IP) step recovers only the tiny fraction of RNA that is actively being transcribed. However, we have optimized protocols for lower inputs (down to 1-5 million cells) for specific sensitive cell lines.
- What is the difference between NET-Seq and PRO-Seq?
  - The main difference is the "Run-On" step. PRO-seq isolates nuclei and "restarts" transcription in a tube using biotin-labeled nucleotides. NET-Seq skips this artificial step and sequences the RNA exactly as it was inside the cell (Native), making it better for studying native pausing kinetics.
- Can you handle immunoprecipitation of specific Pol II modifications?
  - Yes. Our service allows for the use of specific antibodies. While "Total Pol II" (using antibodies like 8WG16) is standard, we can also perform mNET-seq using antibodies against Ser5P (initiation-associated) or Ser2P (elongation-associated) phosphorylation states.
- Does the service include bioinformatics for pausing indices?
  - Yes. Our standard deliverable includes a comprehensive analysis package: raw data (FASTQ), aligned reads (BAM), and a detailed report containing Pausing Indices, Metagene Plots, and gene body coverage heatmaps.
- Is this service suitable for clinical diagnostics?
  - No. This service is for Research Use Only (RUO). The data is intended for basic research and discovery purposes.
- How do you ensure library purity from rRNA?
  - We employ a dual strategy: first, our IP wash conditions are optimized to reduce non-specific binding of ribosomes. Second, during library prep, we use a targeted depletion strategy to remove abundant rRNA sequences.

References:

Nojima T, et al. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell (2015).
Churchman LS, Weissman JS. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature (2011).
Mayer A, et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell (2015).