Alternative Splicing Regulation
Identify RBP binding sites at splice junctions, cassette exons, or intronic regions to understand how RNA-binding proteins modulate splicing outcomes. Combine with RNA-seq for ΔΨ correlation analysis.
iCLIP Service — High-Confidence Mapping of RNA–Protein Interactions
Discover where, how, and why your RNA-binding proteins interact with RNA—at single-nucleotide resolution.
At Creative Proteomics, our expert iCLIP service combines high-precision biochemistry with advanced informatics to uncover regulatory logic, track differential binding, and link RBP occupancy to functional RNA outcomes. Whether you're studying splicing, RNA stability, or disease-associated variants, we help you pinpoint critical RBP–RNA interactions with confidence.
Why Choose Our iCLIP Service:
- Single-Nucleotide Precision — RT-stop–anchored crosslink mapping for accurate site localization
- Optimized Wet Lab Workflow — Low background, high enrichment, and reproducible libraries
- Comprehensive Analysis — Binding site discovery, motif enrichment, and cross-condition comparison
- Biological Insight Delivery — Integrated RNA-seq, APA, splicing, and pathway interpretation
What Is iCLIP?
iCLIP (individual-nucleotide resolution CLIP) uses short-wavelength UV to covalently crosslink endogenous RNA–protein complexes in cells or tissues. After immunoprecipitating the target RNA-binding protein (RBP), reverse transcription terminates at the crosslink site, generating truncated cDNAs that pinpoint binding positions at single-nucleotide resolution. Compared with RIP-seq or standard CLIP, iCLIP delivers superior positional accuracy and more reliable motif discovery, enabling quantitative comparisons across conditions and seamless integration with transcriptome, splicing, and 3'UTR regulation analyses.
Key outputs
- Crosslink sites and peaks with genomic coordinates
- Feature-level binding preferences across introns, exons, UTRs, and splice junctions
- Motif discovery and metagene profiles around crosslink sites
- Differential binding between experimental conditions
- Ranked target lists with pathway and network context
Typical Scientific Questions iCLIP Can Answer
- Where does my RBP bind? Identify base-resolution crosslink sites across exons, introns, UTRs, and non-coding RNAs.
- What motifs or structures are recognized? Reveal enriched sequence or structural elements near binding sites.
- Does binding change across conditions? Compare wild-type vs mutant or control vs treated samples to detect differential binding.
- What does binding regulate? Integrate with RNA-seq to link binding to splicing, stability, or translation.
- Which targets matter most? Rank transcripts by peak strength, motif presence, conservation, and pathway relevance.
- Do mutations alter binding? Assess binding changes caused by RNA or protein variants.
- Can I resolve binding in complex systems? Map RBP behavior in tissues, developmental stages, or stress responses.
Advantages of Our iCLIP Service
Truncation-Anchored Crosslink Site Mapping — Base-Resolution Precision via UV-C Induced RT Stops
Captures reverse transcriptase termination at crosslink sites to pinpoint direct RNA–protein interactions at single-nucleotide resolution with high specificity.
High Signal-to-Noise Workflow — Optimized Lysis, RNase Titration, and Adapter Design
Minimizes non-specific background and preserves authentic binding signals through refined biochemical protocols and library construction strategies.
Reproducible Peak Calling — Replicate Concordance and IDR-Based Confidence Scoring
Quantifies inter-replicate overlap using Irreproducible Discovery Rate (IDR) and models peak robustness for statistically defensible binding site identification.
Motif-Centered Interpretation — De Novo Discovery of RBP Binding Preferences
Discovers sequence and structure motifs enriched near crosslink sites, revealing RBP targeting logic and enabling predictive downstream analyses.
Differential Binding Analytics — Detect Regulatory Rewiring Across Conditions
Identifies condition-specific gains or losses in RBP occupancy across wild-type, mutant, treatment, or stress states using model-based statistical contrasts.
Integrated Multi-Omics Reporting — Link Binding to RNA Abundance and Splicing
Combines iCLIP data with RNA-seq or other transcriptomic layers to uncover mechanisms by which RBPs affect transcript stability, splicing, or translation.
Molecular Interaction Analysis
Service Scope
Workflow and Instrumentation
Sample Requirement
Deliverables
Which to Choose
FAQ
Get a Custom Proposal
Scope of iCLIP Services at Creative Proteomics
3'UTR Binding and RNA Stability
Map binding patterns along 3' untranslated regions (3'UTRs) to infer regulation of mRNA decay, polyadenylation site usage, or miRNA-mediated repression.
Neurogenesis and Developmental Programs
Characterize dynamic RBP–RNA interactions during stem cell differentiation, organogenesis, or neuronal maturation, often with spatiotemporal resolution.
Stress and Environmental Response
Uncover how RBP binding is reprogrammed under stress conditions (e.g., heat shock, hypoxia, oxidative stress) to influence transcript stability and processing.
Host–Pathogen Interactions
Explore RBP recruitment to viral RNA genomes or host regulatory RNAs involved in antiviral defense, RNA sensing, or immune modulation.
Alternative Polyadenylation (APA) Analysis
Analyze binding near APA sites to investigate RBP roles in 3'UTR shortening, isoform diversity, and translational control.
Non-Coding RNA Interactions
Investigate RBP binding to lncRNAs, circRNAs, or pri-miRNAs to uncover non-coding regulatory networks and chromatin–RNA coupling.
Comparative RBP Binding Landscapes
Perform differential iCLIP across conditions, cell types, tissues, or species to identify conserved and rewired RNA targets.
Our iCLIP Service Workflow
1
Project Scoping & Antibody Strategy
Define biological question, select RBP/epitope, plan controls and replicates; verify antibody specificity (or tag strategy).
2
Crosslinking & Immunoprecipitation
UV-C crosslinking (on ice), optimized lysis, limited RNase digestion, IP with validated antibody, stringent washes.
3
On-Bead Processing & Library Prep
3' adapter ligation, reverse transcription with unique barcode incorporation, truncation capture, cDNA circularization/linearization, PCR amplification, and size selection.
4
Sequencing
Illumina PE or SR runs with appropriate read length; lane-level QC and balanced indexing.
5
Computational Pipeline
Demultiplex → UMI deduplication → adaptor/quality trimming → alignment → crosslink event calling → peak models → motif & meta-feature analyses.
6
Biological Interpretation & Delivery
Differential binding vs controls/conditions, integration with RNA-seq/AS/APA, pathway/target impact, figures, and final report.
iCLIP Instrumentation & Technical Capabilities
Crosslinking: UV-C (commonly 254 nm) on cells or pulverized tissue; calibrated energy windows to protect RNA integrity while maximizing crosslinks.
Immunoprecipitation: Magnetic bead systems; high-stringency buffers; support for endogenous or affinity-tagged proteins.
Library Chemistry: Adapter-ligated reverse transcription; cDNA truncation capture; stringent size-selection for iCLIP-characteristic insert sizes.
Sequencing Platforms: Illumina NextSeq/NovaSeq class; typical 8–25+ million processed reads per library (project-dependent).
Bioinformatics Stack: FastQC/MultiQC, cutadapt, STAR/Bowtie2, CLIP-specific peak callers (CITS/CTK-like), MEME-style motif tools, rMATS/MAJIQ for splicing integration, APA quantification, custom R/Python visualization.
Quality Metrics: Library complexity (pre/post-UMI), fraction of reads in peaks (FRiP), crosslink site enrichment at motifs, replicate concordance, insert-size profiles, IP enrichment over controls.
Thermo Fisher DynaMag-2 / DynaMag-15
Agilent 2100 Bioanalyzer
Illumina NextSeq 2000
Sample Requirements for iCLIP Service
| Category | Required Specification (Client to Provide) | Notes / Options |
| Sample type | Cultured cells (suspension/adherent; primary or cell line) or fresh-frozen tissue | FFPE is not accepted |
| Input per IP | Cells: ≥ 1–5 × 106 cells per IP Tissue: ≥ 20–50 mg per IP |
Depends on RBP abundance and antibody performance; low-input option below |
| Project scale | 2–3 biological replicates per condition | Improves statistical power and peak reproducibility |
| Controls | IgG and Input controls; No-UV control if feasible | Essential for specificity/background modeling |
| Crosslinking | UV-C 254 nm; indicate whether samples arrive pre-crosslinked | We can crosslink on receipt or accept client-crosslinked, snap-frozen pellets |
| Sample state & shipping | Cell pellets or tissue chunks (≤ ~50 mg each), shipped on dry ice | RNase-free handling, avoid freeze–thaw; label biosafety level |
| Antibody | IP-grade antibody to target RBP; recommend ≥10–20 µg reserved for the batch | Provide vendor/catalog/lot/host species; tag strategy possible on request |
| Metadata | Species, tissue/cell line, treatment (dose/time), passage/batch, cell viability, expected RBP expression | Used for library setup and bioinformatics grouping |
| Low-input option (optional) | 0.5–1 × 106 cells/IP or ≤20 mg tissue/IP | Requires low-input library chemistry; consider more replicates |
Deliverables: What You Get from Our iCLIP Service
- Raw sequencing files (FASTQ)
- Aligned reads (BAM + index)
- Crosslink sites and binding peaks (BED)
- BED/BEDPE files (binding sites)
- Motif discovery results
- Genome browser tracks
- Normalized binding counts
- Key QC summary (library/sequencing)
Motif Logo with Enrichment
Sequence logo of the iCLIP-enriched motif with site count, E-value, q-value, and odds-ratio enrichment; PWM shown at right.
Genome Browser View of Crosslink Peaks
Representative transcript (e.g., ACTB) with iCLIP crosslink density track, annotated peak region, and motif sites overlaid on the exon–intron gene model, highlighting precise binding in the 3'UTR.
Meta-gene Binding Profile (Aggregate Plot)
Aggregated, normalized crosslink density across 5'UTR, CDS, and 3'UTR; prominent rise near the start codon, dip within CDS, and increase toward the stop codon. Shaded band indicates SEM.
Differential Binding Heatmap (Conditions Comparison)
Heatmap comparing binding across genes between control and treated conditions; colors encode log2FC (blue = down, red = up), with hierarchical clustering and FDR annotations to highlight condition-responsive targets.
iCLIP vs. Other CLIP-Seq and RNA-Binding Techniques: A Side-by-Side Comparison
| Dimension | iCLIP | eCLIP | PAR-CLIP | HITS-CLIP | RIP-seq | CLASH/hiCLIP | irCLIP / low-input | RAP-MS / ChIRP-MS | APEX-Seq |
| Crosslinking / Labeling | UV 254 nm | UV 254 nm + size-matched input | UV 365 nm + 4SU/6SG labeling | UV 254 nm | None (native IP) | UV 254 nm + proximity ligation | UV 254 nm; infrared adapters | Antisense capture; biotin pull-down | Enzyme proximity biotinylation (live) |
| Nucleotide Resolution | Single-nt (truncation sites) | Near single-nt (peak centroids) | Single-nt (T→C / G→A) | Sub-nt to tens of nt | Region-level | Chimeric junctions (RNA–RNA) | Near single-nt | N/A (protein IDs) | Region/proximity |
| Primary Outputs | Site-resolved peaks; motifs | Peaks with background control | Peaks with mutation signature | Peaks across exons/UTRs | Enriched transcripts | RBP-bridged RNA–RNA pairs | Site-resolved peaks from low input | Proteins bound to a chosen RNA | RNAs near tagged protein/compartment |
| Key Strengths | Highest positional precision; motif discovery | Standardized; strong reproducibility | Clear crosslink signature; high S/N | Proven, flexible | Simple; low barriers | Reveals duplex partners | Works with scarce samples | Direct RNA-centric proteome | Live-cell spatial context |
| Main Limitations | Library complexity; needs replicates | Slightly less precise than iCLIP | Requires nucleoside labeling | Lower site precision; background | Indirect/bridged interactions; low positional info | Complex; fewer informative reads | Special adapters/instrumentation | No site maps; probe design critical | Proximity ≠ direct binding; needs tagging |
| Typical Input* | ≥5–10 M cells or 50–200 µg lysate | Similar to iCLIP | Labeled culture; ≥5–10 M cells | Similar to iCLIP | 1–5 M cells (can be lower) | ≥10–20 M cells | ~0.5–1 M cells (context-dependent) | 1–5×10^7 cells | Stable cell lines |
| Best-fit Use Cases | Precise site mapping & motif grammar | Broad discovery with strong controls | Cultured cells where labeling is feasible | Global landscapes w/ moderate precision | Screen/confirm RNA partners (no sites) | Mechanistic RNA–RNA interaction mapping | Primary/limited material | Identify proteins on specific RNA | Spatial transcript proximity to RBPs |
Quick selection guide:
- Need exact binding sites and motifs across the transcriptome → iCLIP (or eCLIP if you want standardized background controls).
- Need diagnostic crosslink mutations and strong S/N in cultured cells → PAR-CLIP (requires 4SU/6SG labeling).
- Have very limited input → irCLIP/low-input CLIP variants.
- Want RNA–RNA interactions mediated by your RBP → CLASH/hiCLIP.
- Just want to know which RNAs are associated (not sites) or have low complexity samples → RIP-seq.
- Want the proteome bound to one specific RNA → RAP-MS/ChIRP-MS.
- Want live-cell spatial proximity of RNAs to a protein/compartment, not necessarily direct binding → APEX-Seq.
You May Want to Know
Which species and reference genomes are supported?
Human, mouse, rat, zebrafish, Drosophila, Arabidopsis, yeast, and custom genomes. We can also work with alternative annotations (GENCODE/RefSeq/Ensembl) or client-provided GTFs.
Can you proceed without a validated antibody?
Yes—options include testing multiple candidate antibodies, using epitope-tagged constructs (e.g., FLAG/HA/V5), or pilot IP-western evaluations to select the best reagent.
Do you support tissue samples and hard-to-lyse materials?
Yes. We provide optimized lysis for neural tissue, heart, and other tough matrices, plus cryo-pulverization when needed. Consult us for buffer compatibility and RNase control.
What if my RBP is lowly expressed?
We can employ low-input iCLIP chemistry, increased replicate counts, and deeper sequencing. UMIs and stringent deduplication help recover true signals from scarce material.
What read configuration works best?
Either SR75–SR100 or PE75–PE100 is typical. The key is sufficient read length to anchor crosslink sites and map uniquely around UTRs and splice junctions.
What background controls do you recommend beyond IgG/Input?
When feasible: no-UV controls, isotype controls, and size-matched input (SMI) libraries to model background and improve specificity of downstream peak filters.
Can you re-analyze existing CLIP or RIP data I already have?
Yes. We can import prior FASTQ/BAM/bedGraph files, harmonize processing with our iCLIP pipeline, and produce comparative reports across datasets and conditions.
Do you support structure-aware or motif-centric analyses?
Yes. We can incorporate secondary structure predictions, RNA accessibility scores, and de novo motif discovery, then quantify motif enrichment at crosslink sites.
Can iCLIP be combined with RNA-seq, APA, or splicing analyses?
Yes. We routinely integrate iCLIP peaks with differential expression, alternative splicing (ΔΨ), and APA metrics to link occupancy with functional outcomes.
How do you ensure reproducible peak calls?
We use replicate-aware modeling and IDR-style concordance assessments, plus FRiP, insert-size profiles, and crosslink-to-motif enrichment as QC gates.
Do you support cross-species or cross-tissue comparisons?
Yes. We can standardize pipelines across species or tissues, liftOver conserved regions when appropriate, and report shared versus rewired binding programs.
What if my protein binds structured or repetitive RNAs?
We apply tailored alignment parameters, multi-mapping strategies, and post-filtering to handle repeats and structured elements while controlling false positives.
Can you prioritize biologically relevant targets?
Yes. We rank peaks/targets using effect size, motif presence, conservation, and pathway/network context, and can deliver shortlists tailored to your hypothesis.
