What is chromatin-associated RNA-seq (CB RNA-seq) and when should I use it?

CB RNA-seq isolates RNA physically bound to chromatin. Use it when you need to measure nascent transcription rates, study co-transcriptional splicing, detect enhancer RNAs (eRNAs), or distinguish between changes in RNA synthesis versus RNA stability.

How is CB RNA-seq different from nuclear RNA-seq or total RNA-seq?

Total RNA-seq measures the steady-state transcriptome. Nuclear RNA-seq includes both chromatin-bound and soluble nuclear RNA. CB RNA-seq strictly analyzes the chromatin-bound fraction, offering the highest resolution for immediate transcriptional events.

What fractionation QC evidence do you provide?

We offer optional Western Blotting for protein markers (H3, GAPDH) and RT-qPCR for RNA markers (Intron/Exon ratios). Standard deliverables include sequencing-based QC metrics such as intronic read fraction and intergenic coverage analysis.

Can CB RNA-seq support low-input or precious samples?

Generally, no. The physical fractionation process involves multiple wash steps resulting in material loss. We recommend a minimum of 5-10 million cells.

Chromatin-bound RNA Sequencing Service

Chromatin-associated RNA sequencing (CB RNA-seq), also known as chromatin-bound RNA-seq, is a specialized transcriptomic approach designed to enrich for nascent, physically chromatin-tethered RNA molecules. Unlike total RNA-seq or nuclear RNA-seq, which capture a mixture of processing intermediates and mature stable transcripts, CB RNA-seq strictly isolates the RNA fraction associated with the chromatin complex.

Key Highlights:

Capture nascent transcripts and intronic reads to measure immediate transcription rates.
Decouple transcriptional synthesis from RNA stability and decay kinetics.
Map active enhancers (eRNAs) and super-enhancers with high sensitivity.
Validated biochemical fractionation with strict QC for nuclear/cytoplasmic purity.

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$Workflow of chromatin-associated RNA-seq (CB RNA-seq) service from fractionation to bioinformatics deliverables.$

Overview: Capturing nascent transcription measures

This service is engineered for study designs requiring the decoupling of transcriptional synthesis rates from RNA decay kinetics. By sequencing the chromatin fraction, you access a snapshot of the genome in action—capturing unspliced pre-mRNAs and chromatin-enriched RNAs (cheRNAs) before they are released into the nucleoplasm or exported to the cytoplasm.

Our end-to-end workflow incorporates rigorous subcellular fractionation protocols, validated by compartment-specific quality control (QC) markers, ensuring that the sequencing data reflects true transcriptional activity rather than background noise from stable cytoplasmic pools. This approach is particularly powerful for identifying immediate-early response genes, mapping active enhancers, and characterizing the direct effects of therapeutic compounds on transcriptional machinery.

It is important to clarify what this service does not measure: CB RNA-seq is not a proxy for steady-state gene expression levels, which are better assessed via total RNA-seq. Furthermore, while it enriches for nascent RNA, it is distinct from metabolic labeling methods (like PRO-seq) that measure polymerase position; instead, it measures the accumulation of RNA physically tethered to chromatin.

Method Selection: CB RNA-seq vs. PRO-seq/RPRO-seq

Choosing the right nascent RNA profiling method depends on whether your research question focuses on the accumulation of chromatin-tethered transcripts or the active position of RNA Polymerase II.

Feature	Chromatin-Bound (CB) RNA-seq	PRO-seq / RPRO-seq
Principle	Physical biochemical fractionation of the chromatin complex.	Nuclear run-on with biotinylated NTPs to map active RNA Polymerases.
What it measures	RNA molecules currently tethered to chromatin (nascent pre-mRNA + chromatin-retained lncRNAs + eRNAs).	Density of active polymerases across the genome at single-nucleotide resolution.
Key Advantage	Chemical-free (no exogenous nucleotides), simpler workflow, captures full transcript length.	Maps polymerase pausing (traveling ratio) and immediate TSS with higher resolution.
Best for	Co-transcriptional splicing, chromatin retention of lncRNAs, nascent gene expression.	High-resolution polymerase mapping and pausing dynamics.
Our Services	Chromatin-bound RNA Sequencing Service	View PRO-seq Service or RPRO-seq (Low Input)

Applications & study designs for mechanism discovery

Decoupling Transcription from Stability

Standard transcriptomics cannot distinguish between increased RNA synthesis and decreased RNA degradation. CB RNA-seq resolves this by profiling the "production center" of the cell. High levels of intronic reads in the chromatin fraction serve as a direct proxy for transcription rates.

Enhancer RNA (eRNA) Detection

Enhancer RNAs are typically unstable and low-abundance. Because eRNAs function locally on chromatin, they are significantly enriched in the chromatin-bound fraction. Our service allows for high-sensitivity mapping of active enhancers and super-enhancers.

Co-transcriptional Splicing Dynamics

By capturing RNA molecules that are still tethered to the DNA template, CB RNA-seq provides a window into co-transcriptional splicing. Researchers can analyze the ratio of unspliced to spliced reads across the gene body to study splicing kinetics.

Immediate-Early Gene (IEG) Response

Chromatin fractionation removes stable RNA background, significantly increasing the signal-to-noise ratio for detecting rapid transcriptional changes within minutes of stimulation (e.g., cytokine signaling, neuronal activation).

Non-coding RNA Chromatin Occupancy

CB RNA-seq quantitatively assesses the chromatin enrichment of specific lncRNAs compared to nucleoplasmic or cytoplasmic fractions, supporting hypotheses regarding their roles in epigenetic regulation or chromatin remodeling.

Wet-lab workflow (fractionation → library prep)

Our wet-lab process is optimized to maintain RNA integrity during the rigorous physical separation of cellular compartments.

Phase 1: Stepwise Subcellular Fractionation – We utilize a detergent-based biochemical fractionation protocol. Cells are lysed to remove cytoplasm, nuclei are washed and extracted to remove nucleoplasm, and the final chromatin pellet is solubilized to release tightly bound RNAs.
Phase 2: RNA Extraction and rRNA Depletion – RNA is extracted from the protein-rich chromatin pellet. We employ a rigorous rRNA depletion step targeting both cytoplasmic and mitochondrial rRNA, as poly-A selection is unsuitable for nascent transcripts.
Phase 3: Stranded Library Preparation – Strand-specific library preparation (dUTP-based) is mandatory to correctly assign reads to sense (coding) or antisense (regulatory) strands.
Final QC – Libraries are assessed for size distribution and concentration before pooling for sequencing.

$End-to-end CB RNA-seq workflow from fractionation to analysis-ready outputs.$

Fractionation quality & QC evidence

The validity of CB RNA-seq data hinges entirely on the purity of the fractionation. We provide a multi-tiered QC package to validate fractionation efficiency.

1. Western Blot Validation

Optional add-on. We test for Chromatin Markers (Histone H3, RNA Pol II) and Cytoplasmic Markers (GAPDH, Tubulin) to ensure successful isolation and lack of contamination.

2. RT-qPCR Marker Analysis

We measure Intron/Exon ratios for housekeeping genes. The chromatin fraction should show a significantly higher Intron/Exon ratio compared to the cytoplasm.

3. Sequencing-Based QC

Post-sequencing metrics confirm library nature: 20%–50% intronic reads, elevated intergenic coverage (eRNAs), and uniform gene body coverage.

Bioinformatics deliverables

Our bioinformatics pipeline is customized to handle the unique characteristics of chromatin-associated RNA-seq data, including intronic read retention and eRNA identification.

Standard Analysis Module

Raw Data QC & Alignment (Splice-aware)
Full-Gene Counts (Exon + Intron): Essential for quantifying total transcriptional output.
Differential Expression Analysis (DESeq2/edgeR) on nascent transcripts.

Advanced Mechanism Analysis Module

Intron/Exon Ratio Analysis: Proxy for splicing efficiency or transcriptional progression.
Metagene Profiling: Visualizing polymerase distribution and potential pausing.
eRNA Identification: Calling peaks in intergenic regions/enhancers.
Visualization Files: BigWigs normalized by depth for genome browser viewing.

Sample requirements

High-purity fractionation requires sufficient starting material. Please adhere to the following input guidelines.

Sample Type	Minimum Input	Recommended Input	Collection & Preservation
Adherent Cells	5 x 10⁶ cells	1 x 10⁷ cells	Wash in PBS, pellet, flash freeze dry. Viability >90% required.
Suspension Cells	5 x 10⁶ cells	1 x 10⁷ cells	Wash, pellet, remove all supernatant, flash freeze. No RNALater.
Soft Tissue	20 mg	50 mg	Resect and flash freeze in liquid nitrogen within 2 mins. (Liver/Brain/Kidney).
Fibrous Tissue	50 mg	100 mg	Flash freeze. Requires specialized pulverization (cryo-grinding).

Note: For all samples, please include a manifest detailing cell type and estimated count. If requesting Western Blot QC, indicate if primary antibodies will be provided.

Case Study: Resolving Transcriptional Pausing

Background

A research group investigating a novel CDK9 inhibitor needed to determine if the drug caused global transcriptional shut-off or specifically increased promoter-proximal pausing. Total RNA-seq showed downregulated gene expression but could not resolve the kinetic mechanism.

Methodology

Chromatin-associated RNA-seq was performed on treated vs. control cells to isolate the nascent transcriptome. As detailed in robust protocols (Pandya-Jones et al., 2022), this fractionation approach effectively separates chromatin-tethered RNAs from the nucleoplasmic pool, enabling precise mapping of polymerase location.

Results & Conclusion

The analysis revealed a "traveling ratio" shift. While gene body reads decreased, reads at the Transcription Start Site (TSS) remained high, indicating that the polymerase was initiated but failed to elongate. This proved the drug's mechanism as an elongation inhibitor, a conclusion impossible to reach with standard transcriptomics.

Chromatin-associated RNA-seq data showing traveling ratio shift and transcriptional pausing

FAQs – Frequently Asked Questions

- What is chromatin-associated RNA-seq (CB RNA-seq) and when should I use it?
  - CB RNA-seq isolates RNA physically bound to chromatin. Use it when you need to measure nascent transcription rates, study co-transcriptional splicing, detect enhancer RNAs (eRNAs), or distinguish between changes in RNA synthesis versus RNA stability.
- How is CB RNA-seq different from nuclear RNA-seq or total RNA-seq?
  - Total RNA-seq measures the steady-state transcriptome. Nuclear RNA-seq includes both chromatin-bound and soluble nuclear RNA. CB RNA-seq strictly analyzes the chromatin-bound fraction, offering the highest resolution for immediate transcriptional events.
- What fractionation QC evidence do you provide?
  - We offer optional Western Blotting for protein markers (H3, GAPDH) and RT-qPCR for RNA markers (Intron/Exon ratios). Standard deliverables include sequencing-based QC metrics such as intronic read fraction and intergenic coverage analysis.
- Can CB RNA-seq support low-input or precious samples?
  - Generally, no. The physical fractionation process involves multiple wash steps resulting in material loss. We recommend a minimum of 5-10 million cells. For low-input applications, consider alternative methods like CUT&Tag-based RNA detection.
- Can CB RNA-seq help distinguish transcriptional regulation from RNA stability effects?
  - Yes. Since chromatin-bound RNA represents newly synthesized transcripts, changes in this fraction reflect transcriptional regulation. If Total RNA changes but Chromatin RNA does not, the effect is likely post-transcriptional (stability).
- How do you interpret enhancer RNA (eRNA) signals?
  - eRNAs appear as low-abundance, often bidirectional transcripts in intergenic regions. By mapping these reads to known enhancer regions (defined by H3K27ac), we can quantify enhancer activity cleanly, which is often masked in total RNA data.

References:

Pandya-Jones, A., et al. A simple and robust method for isolating and analyzing chromatin-bound RNAs. SpringerOpen, 2022.
Bell, J.C., et al. Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts. eLife, 2018.
Werner, M.S., et al. Mapping Transcriptome-Wide and Genome-Wide RNA–DNA Interactions Using ChAR-seq. Springer Protocol, 2020.
Nojima, T., et al. Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells. Springer Protocol, 2024.
Bhatt, D.M., et al. Protocol for quantitative analysis using chromatin-associated RNA-seq data. ScienceDirect, 2023.