PIRCh-seq: Unlock RNA-Chromatin Interactions for Gene Regulation Insights
Understanding how non-coding RNA interacts with chromatin is key to unlocking gene expression regulation. CD Genomics' PIRCh-seq technology offers a precise and high-throughput solution to explore these critical RNA-chromatin interactions. This innovative approach allows you to gain deep insights into RNA regulation under chromatin's epigenetic modifications, paving the way for breakthrough discoveries in gene regulation and epigenetics.
Key Benefits:
In-Depth RNA-Chromatin Interaction Insights: Uncover how RNA influences gene expression through chromatin modifications.
High-Throughput Data: Achieve comprehensive, high-quality data from a single experiment, maximizing research efficiency.
Reliable Data Accuracy: Leveraging high specificity antibodies, PIRCh-seq ensures accurate and reproducible results.
PIRCh-seq detects RNA-chromatin interactions genome-wide by integrating RNA immunoprecipitation with chromatin profiling. This method captures non-coding RNAs bound to epigenetically modified chromatin regions, particularly histone marks associated with active or repressed transcriptional states.
Low-abundance RNAs require ≥10 million cells per replicate.
PIRCh-seq Workflow: Three Steps to Unlock RNA-Chromatin Interactions
PIRCh-seq is a cutting-edge technique for studying the interactions between non-coding RNAs (like lncRNAs, miRNAs) and chromatin modifications. The workflow focuses on preserving authentic interactions and enhancing data precision. From cell handling to data analysis, each step addresses the common challenges faced in traditional immunoprecipitation methods, such as RNA degradation and non-specific binding.
Chromatin Fragmentation: Chromatin is fragmented using ultrasonic disruption, creating fragments ranging from 300-2000bp, balancing yield and integrity.
Antibody Selection Strategy:
For Activation Zone RNA: Antibodies like H3K4me3, H3K27ac are used.
For Repression Zone RNA: Antibodies such as H3K27me3, H3K9me3.
For RNP-Mediated Interactions: Antibodies like Ago2, EZH2 are employed.
Decontamination Process:
DNA Digestion: DNase I is used to eliminate residual DNA.
Protein Digestion: Proteinase K digests binding proteins.
Output: High-purity chromatin-bound RNA, with an OD260/280 ratio of 1.8-2.0, ensuring high-quality samples for analysis.
3. Interaction Mapping: From Sequence to Regulatory Logic
Library Construction Strategy:
For Long RNA: lncRNA libraries that retain full-length sequences.
For Short RNA: Small RNA libraries that capture miRNAs and other small RNAs.
The resulting data is analyzed to provide a comprehensive map of RNA-chromatin interactions, enabling researchers to pinpoint functional chromatin regions and understand the regulatory roles of RNA in gene expression.
Applications
PIRCh-seq's Four Core Applications
1. Mapping the "Histone Modification – Non-Coding RNA" Interaction Landscape
Challenge: Researchers often suspect a lncRNA's role in gene silencing but are uncertain where it binds on chromatin.
PIRCh-seq Advantage: By using modification-specific antibodies (like H3K4me3, H3K27ac), PIRCh-seq directly captures:
lncRNAs binding to active promoter regions (e.g., PVT1 regulating MYC).
RNA silencing in heterochromatin regions (e.g., XIST binding to H3K27me3).
Output: PIRCh-seq provides a three-tier evidence chain—epigenetic modification state, RNA location, and target gene regulation—avoiding the ambiguity seen in traditional technologies that only show "RNA on chromatin."
Research Dilemma: While it's known that RNA-binding proteins like Ago2 participate in gene regulation, the exact mechanism of RNA-chromatin bridging remains unclear.
Whether RBPs recruit specific RNAs to chromatin (e.g., Ago2-mediated miRNA targeting to enhancers).
The distribution hotspots of RBP-RNA complexes across the genome (e.g., telomeric and centromeric regions).
PIRCh-seq vs. CLIP: Unlike CLIP, PIRCh-seq captures both the RBP-RNA binding sites and the chromatin environment at these sites (e.g., whether they reside within super-enhancers).
3. Discovering "Hidden Regulators" in Enhancer Regions: Nuclear miRNAs
Knowledge Gap: Historically, miRNAs were thought to function only in the cytoplasm. However, recent studies show nuclear miRNAs can bind to enhancers and regulate transcription.
PIRCh-seq Advantage: Through a genome-wide scan, PIRCh-seq identifies:
Nuclear miRNAs bound to enhancer regions (e.g., H3K27ac-marked areas).
Their targeted neighboring genes (e.g., oncogene promoters).
4. Decoding the Impact of RNA Modifications (e.g., m6A) on Chromatin Localization
Scientific Question: Does RNA modification like m6A serve as a "postal code" to determine its chromatin destination?
PIRCh-seq Advantage: PIRCh-seq compares the chromatin distribution of m6A-modified RNA versus unmodified RNA, identifying:
Regions enriched with modified RNA (e.g., m6A-marked lncRNAs in transcription initiation regions).
Experimental Design Suggestion: Combine PIRCh-seq with MeRIP-seq (m6A profiling) to validate causal relationships between RNA modifications and their chromatin locations.
RNA-Chromatin Research: A Quick Overview of 4 Key Technology Differences
The Only Technology to Analyze Both RNA Identity and Epigenetic Localization
PIRCh-seq offers an unparalleled depth of analysis in RNA-chromatin interactions by examining both the RNA identity and its precise epigenetic location. Here's a comparison of PIRCh-seq with other core technologies:
Technology
Core Capability
Limitations
Choose PIRCh-seq If You Need →
RIP
Identifies RNA-protein interactions
Cannot pinpoint chromatin binding sites
→ Know where RNA interacts with specific epigenetic regions
CLIP
Analyzes RNA-protein binding sequences
Cannot associate with chromatin functional regions
Conventional RNA-chromatin interaction detection methods (such as ChIRP-seq) can only answer the question of whether RNA binds to chromatin, but they cannot distinguish the epigenetic modification context in which the RNA binds (e.g., activating H3K27ac vs. repressive H3K27me3).
PIRCh-seq's Breakthrough
PIRCh-seq overcomes this limitation by using histone modification-specific antibodies (such as H3K4me3, H3K27ac), directly locking in:
RNA's specific location in functional chromatin regions (e.g., promoters, enhancers).
How the epigenetic state of the region affects RNA functionality (e.g., lncRNA activating transcription in H3K4me3 regions).
2. Dual-Dimensional RNA Capture Ability
PIRCh-seq excels in covering two key regulatory molecules simultaneously:
Long non-coding RNAs (lncRNAs): Investigate their role in recruiting chromatin-modifying complexes.
Small RNAs (miRNAs/snoRNAs): Analyze their regulatory role at enhancer and other chromatin elements.
Note: Unlike traditional methods that require separate library construction, PIRCh-seq allows for synchronous capture in a single experiment, significantly improving efficiency and data integration.
3. Innovative Interaction Preservation Technology
Solving Crosslinking Damage Issues
PIRCh-seq uses glutaraldehyde crosslinking combined with glycine quenching (non-UV crosslinking) to preserve the natural structure of RNA-chromatin-protein complexes, avoiding the degradation and loss associated with traditional methods.
Optimized Fragmentation Range
PIRCh-seq uses an optimized ultrasonic fragmentation range (300-2000bp), which prevents lncRNA fragmentation, a common issue with smaller fragments in other methods.
4. Cross-Species Compatibility Design
Validated Species
PIRCh-seq has been successfully validated for use with species such as humans, mice, and rats, where complete reference genomes are available.
Antibody Conservancy Assessment
The adaptability of PIRCh-seq for other species is evaluated based on antibody conservancy (such as for histone modification antibodies).
Pre-Analysis for Reference Genome Compatibility
CD Genomics offers pre-analysis to assess the compatibility of reference genomes, ensuring that the chosen species is suitable for PIRCh-seq-based RNA-chromatin interaction studies.
Service Workflow
PIRCh-seq Service Workflow
Bioinformatics
PIRCh-seq Data Analysis: Rigorous Workflow and Biological Interpretation
Ensuring results are interpretable, verifiable, and extendable, PIRCh-seq's data analysis is designed to offer clear insights into RNA-chromatin interactions. Here's a breakdown of the key analysis steps:
1. Data Quality Control and Standardization
Data Cleaning: Filtering out low-quality sequences and adapter contamination ensures only reliable data is used.
Sequence Alignment:
Reads are mapped to a reference genome following standardized processes.
Alignment efficiency and strand specificity are assessed to ensure accurate data.
Enrichment Verification:
Signal-to-noise ratio of target regions (e.g., antibody-binding sites) is carefully checked to confirm efficient RNA enrichment.
2. Non-Coding RNA Functional Analysis
Annotation and Classification:
Identifies RNA types binding to chromatin, such as lncRNA, miRNA, etc.
Annotates their genomic locations and associated nearby genes.
Differential Analysis:
Compares RNA binding intensities between different samples, identifying statistically significant differences.
Functional Association:
Predicts regulatory mechanisms based on the epigenetic modification states (e.g., H3K27ac correlates with activation).
3. Pathway and Mechanism Exploration
Target Gene Function Enrichment:
Conducts GO/KEGG analysis for genes near differentially bound RNA (±100kb).
Focuses on pathways related to chromatin regulation, such as transcription factor binding and nucleosome remodeling.
Interaction Network Construction:
Integrates RNA location and epigenetic modification states to generate regulatory hypotheses for deeper biological insights.
4. Customizable Report Framework
Each report is tailored to your research needs, ensuring a comprehensive view of the data:
Module
Description
Data Quality Summary
QC metrics and reliability evaluation
Differential RNA List
Includes genomic coordinates and associated genes
Functional Enrichment
Significantly enriched pathways and potential mechanisms
