Chromatin Isolation by RNA Purification Followed by Sequencing (ChIRP-seq): Principles, Applications, and Technical Advances

Chromatin Isolation by RNA Purification followed by sequencing (ChIRP-seq) has reshaped how we study RNA–chromatin interactions. By enabling direct, genome-wide mapping of RNA occupancy in vivo, ChIRP-seq connects non-coding RNA expression to chromatin-based gene regulation in a way that earlier methods could not.

This article provides a technical overview of ChIRP-seq—covering core principles, typical applications, advantages, limitations, and recent innovations. If you are planning an actual project and need step-by-step guidance on design and analysis, consider pairing this overview with:

• First-Time ChIRP-seq Projects: 7 Common Pitfalls to Avoid
• Odd/Even Probe Design for ChIRP-seq
• ChIRP-seq Experimental Design and Controls

Introduction

The eukaryotic genome is packaged into chromatin, a dynamic complex of DNA, histones, and associated proteins that underpins almost all nuclear processes. Over the past two decades, long non-coding RNAs (lncRNAs) have emerged as key regulators of chromatin remodeling, histone modification, three-dimensional genome organization, and transcription.

Classical approaches—RNA immunoprecipitation, pull-down assays, and loss-of-function studies—provided important clues but mostly yielded indirect or low-resolution information. They struggled to distinguish direct chromatin binding from secondary effects.

The development of Chromatin Isolation by RNA Purification (ChIRP) addressed this gap. By combining tiling biotinylated DNA probes with streptavidin-based pulldown, ChIRP directly isolates chromatin regions bound by a specific RNA. Coupling this with next-generation sequencing (ChIRP-seq) enables unbiased, genome-wide mapping of RNA–genome interactions at high resolution.

Principle of ChIRP-seq

At its core, ChIRP-seq relies on three principles:

In vivo crosslinking of native complexes

Formaldehyde (and optionally UV) crosslinking stabilizes RNA–protein–DNA assemblies inside intact cells, preserving native RNA–chromatin contacts at the moment of fixation.

Sequence-specific capture of the target RNA

A pool of biotinylated antisense oligonucleotides, tiling across the target RNA, hybridizes to the crosslinked RNA under stringent conditions. This provides specificity at the level of RNA sequence rather than protein epitope.

Biochemical isolation of RNA-anchored chromatin

Streptavidin magnetic beads recover the probe–RNA complexes together with associated chromatin and RNA-binding proteins. After elution, the DNA fraction is sequenced to map binding sites (ChIRP-seq), and the protein fraction can be analyzed by mass spectrometry (ChIRP-MS).

This design allows ChIRP-seq to directly profile RNA-centered chromatin occupancy across the genome, without prior assumptions about DNA motifs or target loci.

From Pulldown to Genome-Wide Maps

Once RNA-anchored chromatin is isolated, the ChIRP-seq workflow proceeds as follows:

• Library preparation
  DNA fragments recovered from the pulldown are purified and converted into sequencing libraries.
• High-throughput sequencing
  Libraries are sequenced to generate genome-aligned reads.
• Peak calling
  Reads are mapped to a reference genome, and peak callers such as MACS2 are used to identify enriched regions representing candidate RNA binding sites.
• Functional annotation and integration
  Peaks are annotated relative to genes, promoters, enhancers, and other regulatory elements, then integrated with orthogonal datasets (e.g., ChIP-seq, ATAC-seq, RNA-seq) to place RNA occupancy into a broader regulatory context.

Applications of ChIRP-seq

Functional Genomics: Mechanistic Mapping of lncRNAs and circRNAs with ChIRP-seq

One of the earliest and most influential applications of ChIRP-seq was the study of the Drosophila lncRNA roX2, a key component of the Male-Specific Lethal (MSL) complex responsible for X-chromosome dosage compensation. Using ChIRP-seq, Chu (Chu et al., 2011) generated the first base-resolution genome-wide occupancy map of an lncRNA. They demonstrated that roX2 binds specifically to GA-rich Chromatin Entry Sites (CES) on the X chromosome, with a pronounced 3' bias along gene bodies. This binding pattern correlated strongly with MSL localization (Pearson *r* = 0.77) and H4K16 acetylation, a hallmark of active transcription.

Notably, roX2 depletion disrupted MSL recruitment and reduced H4K16ac levels, impairing dosage compensation. These findings revealed that roX2 acts as a sequence-specific guide RNA, directing chromatin-modifying complexes to precise genomic locations—a paradigm shift from the earlier view of lncRNAs as passive scaffolds.

ChIRP-seq Reveals roX2 lncRNA Guides MSL Complex to X-Chromosome CES Sites (Chu et al., 2011)

RNA–Protein Interactomes: Defining RNA-Binding Proteins by ChIRP-MS

A recent ChIRP-seq study of the long non-coding RNA Ppp1r1b-lncRNA illustrates how RNA–chromatin occupancy maps can reveal regulatory mechanisms in development. Working in a mouse muscle myoblast cell line, Hwang and colleagues applied unbiased chromatin isolation by RNA purification coupled to high-throughput sequencing to profile genome-wide Ppp1r1b-lncRNA binding sites.

ChIRP-seq identified tens of thousands of high-confidence peaks distributed broadly across coding and non-coding regions, with nearly half of the binding sites mapping to annotated gene elements and a substantial fraction falling in introns enriched for homeobox-related and TA-rich motifs. These occupancy patterns pointed to a preference for regulatory regions linked to myogenic transcriptional programs.

By integrating ChIRP-seq with chromatin and expression data, the authors showed that Ppp1r1b-lncRNA is recruited to promoters and distal regulatory elements of key myogenic transcription factors, including Tbx5 and MyoD1, where it modulates Polycomb repressive complex 2 (PRC2) activity and H3K27 methylation. This work demonstrates how ChIRP-seq can move beyond simple binding catalogs to uncover lncRNA-directed epigenetic regulation at specific gene networks, in this case promoting cardiac and skeletal muscle development.

Schematic Summary of Ppp1r1b-lncRNA-ChIRP Assay and Bioinformatic Pipeline (Hwang, John Hojoon, et al., 2023).

Disease Mechanisms and Translation: Clinical Applications of ChIRP-seq

ChIRP-seq has also proven valuable in elucidating the role of ncRNAs in human diseases. In a 2025 study published in Nature Communications, Li (Li et al., 2025) used ChIRP-seq to map the genomic binding sites of the renal-specific lncRNA RSDR. They found that RSDR is enriched at the promoter of DHODH, a gene involved in ferroptosis suppression. Integrated ChIRP-seq/MS analysis identified hnRNPK as a direct binding partner, forming an RSDR/hnRNPK/DHODH regulatory axis that inhibits lipid peroxidation and protects against acute kidney injury (AKI).

Translational analysis further revealed that urinary RSDR levels are significantly reduced in AKI patients and exhibit diagnostic potential (AUC = 0.864). This work highlights how ChIRP-seq can uncover novel lncRNA-mediated pathways with clinical relevance, offering new avenues for biomarker discovery and therapeutic targeting.

ChIRP-seq Defines an RSDR/hnRNPK/DHODH Axis Protecting Against Ferroptosis in Acute Kidney Injury (Li et al., 2025)

Technical Advances in ChIRP-seq

Enhanced Specificity and Sensitivity

Several recent improvements have significantly boosted ChIRP-seq performance:

• Probe chemistry
  Incorporation of LNA-modified probes improves hybridization specificity, particularly for GC-rich or structurally complex RNA regions.
• Optimized crosslinking
  Combining formaldehyde with UV crosslinking helps preserve direct RNA–protein contacts while maintaining DNA–protein interactions.
• Improved normalization and peak calling
  New computational methods correct for input variability and probe-specific biases. Supervised machine-learning approaches enhance the discrimination between true peaks and background, especially in complex genomic regions.

These advances collectively expand the method's applicability to low-abundance lncRNAs and more challenging sample types.

For a more design-oriented discussion of low-abundance transcripts and how to plan feasible, high-sensitivity ChIRP-seq experiments, see the sections on expression thresholds and controls in: ChIRP-seq Experimental Design and Controls.

Integration with Emerging Technologies

ChIRP-seq is highly compatible with other cutting-edge technologies:

Chromatin Isolation by RNA Puri…

• CRISPR-based perturbation screens
  Used to functionally validate RNA-bound loci at scale and connect biochemical occupancy with phenotypic consequences.
• Single-cell and spatial omics
  Although technically challenging, adapting ChIRP-like strategies to single-cell or spatial resolution (scChIRP-seq, spatial ChIRP) promises cell-type-specific maps of RNA–chromatin interactions.
• Protocol variants
  Crosslinking-free or RNA–RNA-focused adaptations further broaden the scope of questions that can be addressed.

For a forward-looking discussion of how ChIRP-seq may combine with single-cell and spatial omics in research use, see: ChIRP-seq with Single-Cell and Spatial Omics: Prospects and Design Ideas.

Advantages and Limitations of ChIRP-seq

As an RNA-centered chromatin profiling method, ChIRP-seq offers several distinctive strengths but also carries technical and interpretational caveats that should be considered at the project design stage.

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