eCLIP-Seq Analysis Service

Get reliable, near-base maps of RNA–protein binding that you can trust for decisions and publication. Creative Proteomics delivers end-to-end eCLIP-Seq— from control-aware lab work to transparent, reproducible analysis—so you move from raw reads to defensible biological insight.

  • Near-base binding maps with SMInput + UMI to suppress background
  • Reproducibility built-in (IDR-validated peak sets and site calls)
  • Antibody support (use yours or request screening to de-risk IP)
  • Differential binding, motif & pathway insights in one report
  • Multi-omics options: eCLIP integrated with RNA-seq/splicing/Ribo-seq

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What Is eCLIP-Seq?

eCLIP-Seq (enhanced Crosslinking and Immunoprecipitation Sequencing) is a transcriptome-wide method for mapping in vivo RNA–protein interactions with near-base resolution. The assay combines UV 254 nm crosslinking, antibody-based immunoprecipitation (IP) of the target RNA-binding protein (RBP), and high-throughput sequencing of crosslinked RNA fragments.

Key technical principles that define eCLIP-Seq:

  • SMInput (size-matched input) control to correct for transcript abundance and fragmentation background during peak calling.
  • UMI-aware library construction (8–12 bp) to collapse PCR duplicates and enable molecule-level quantification.
  • Crosslink-derived signatures (read truncations/mismatches) that sharpen nucleotide-scale site localization.
  • Replicate-aware statistics (e.g., IDR) to quantify reproducibility and reduce false positives.

Typical Scientific Questions eCLIP-Seq Can Answer

  • Where does my RBP bind across the transcriptome, and what are the high-confidence peaks and crosslink sites?
  • Is binding enriched in specific features (5′UTR, CDS, 3′UTR, introns) or near splice junctions/branch points?
  • Which sequence or structural motifs are characteristic of the bound regions, and do they match known RBP preferences?
  • How does binding change across conditions (treatment, stress, time course) or between wild type and engineered variants?
  • Are lncRNAs or small RNAs among the direct targets, and how do these contacts relate to RNP assembly pathways?
  • Do binding patterns align with alternative splicing, mRNA stability, or translation differences observed in companion datasets?
  • What off-target RNA interactions are captured by research-grade engineered RBPs or fusion constructs?

Advantages of Our eCLIP-Seq Service

High-Specificity Binding Profiles — SMInput Normalization + UMI Quantification

Each assay incorporates size-matched input controls and 8–12 bp unique tags to reduce background and distinguish PCR duplicates, achieving over 90% fewer false-positive peaks than enrichment-only methods.

Replicate-Robust Discovery — IDR-Driven Confidence

Our analysis applies IDR (Irreproducible Discovery Rate) filtering across biological replicates, ensuring that >80% of reported peaks are reproducible and statistically supported.

Near-Base Resolution — Crosslink-Derived Signatures

We retain crosslink-induced truncations and mismatch events in analysis, enabling nucleotide-level localization of binding sites rather than broad regional footprints.

Optimized Sequencing Depth — Data That Matches Biology

Standard sequencing delivers 25–60 million paired-end reads per IP, scalable up to 100M reads for challenging low-abundance RBPs. This ensures binding maps are deep enough to answer mechanistic questions without over-sequencing.

Sensitive Detection of Low-Abundance RBPs — Optimized IP Chemistry

Enhanced immunoprecipitation and stringent wash protocols improve recovery of rare RBPs, providing reliable maps without relying solely on extreme sequencing depth.

Antibody Validation Support — Ensuring IP Success

For challenging RBPs, Creative Proteomics offers antibody screening or client-supplied validation testing, reducing the risk of failed IPs. This improves first-pass success rate to >85% compared with non-validated antibodies.

Technical Services
Service Scope Workflow and Instrumentation Sample Requirement Deliverables FAQ Get a Custom Proposal

Scope of eCLIP-Seq Services at Creative Proteomics

Splicing-Regulation Mapping — Exon/Junction-Proximal Sites

Best for: RBPs implicated in alternative splicing.

Inputs: Crosslinked cells or tissues; validated antibody; matched input control.

Outputs: Junction- and branch-point–proximal binding maps with motif and feature-level patterns.

Key enablers: Control-aware calling; replicate-aware filtering; crosslink-guided localization.

3′UTR Stability & Translation Control — UTR-Centric Binding

Best for: Post-transcriptional regulation (AREs, poly(A)-proximal regions, miRNA co-occupancy).

Inputs: RBP IP with matched input; optional companion expression/translation dataset.

Outputs: 3′UTR-focused binding landscapes with motif/context summaries and trend correlations.

Key enablers: Crosslink-derived signatures; standardized annotation layers.

Condition-Responsive Binding Dynamics — Differential Occupancy

Best for: Treatment, stress, time-course contrasts; perturbation studies.

Inputs: Replicated IP/input pairs per condition.

Outputs: Ranked differential binding calls with effect sizes and confidence levels.

Key enablers: Harmonized pipelines; replicate-aware statistical modeling; consistent peak universes.

Variant/Domain-Mutant Impact Mapping — Mechanistic Dissection

Best for: WT vs. domain deletions/point mutants or engineered variants.

Inputs: Parallel eCLIP-Seq on isogenic lines or constructs.

Outputs: Gain/loss binding landscapes; domain-dependent motif/context shifts.

Key enablers: Side-by-side processing; IDR-qualified contrasts; motif shift analysis.

Noncoding RNA & RNP Assembly Profiling — lncRNA/sn(o)RNA Engagement

Best for: RNP assembly and ncRNA interaction landscapes.

Inputs: Crosslinked material; antibody validated for the RNP component of interest.

Outputs: Direct contacts on lncRNAs and small RNAs; pathway/complex-level summaries.

Key enablers: Multimapper-aware alignment; targeted ncRNA annotation tracks.

Matched Multi-Omics Context — eCLIP + RNA-seq/Splicing/Ribo-seq

Positioning: Same-source, matched assays to minimize cross-sample variance.

Inputs: eCLIP-Seq with optional RNA-seq (expression), splicing analysis, or Ribo-seq (translation).

Outputs: Integrated hypotheses linking binding events to expression, splicing, or translation changes.

Our eCLIP-Seq Service Workflow

Workflow for eCLIP-Seq
1

Consultation & Panel Design

Target RBP(s), cell/tissue matrix, controls (SMInput, IgG), and replicate scheme. Power and depth recommendations are included.

2

Sample Intake & Qualification

Pre-QC for biomass, RNA integrity proxies, antibody suitability (WB/IP evidence), and crosslinkability.

3

Crosslinking & IP

UV 254 nm crosslink, partial RNase I digest, high-stringency washes, size selection of RBP–RNA complexes.

4

Library Construction

UMI-tagged adapters, reverse transcription capturing crosslink-induced truncations, limited-cycle PCR, library QC.

5

Sequencing

Illumina paired-end 2×75 bp (other formats available on request). Lane loading tuned to target complexity.

6

Bioinformatics & Statistics

UMI-aware processing, normalized peak calling, replicate concordance (IDR), motif/pathway annotation, and optional differential binding.

eCLIP-Seq Instrumentation & Technical Capabilities

Crosslinking: Calibrated UV 254 nm systems with fluence mapping; uniformity documented per batch.

IP & Cleanup: High-capacity magnetic platforms; low-bind plastics validated for minimal RNA adsorption.

Library QC: Qubit fluorometry; fragment analysis via Bioanalyzer/TapeStation; qPCR library quantification.

Sequencing: Illumina NovaSeq / NextSeq platforms; standard 2×75 bp; optional 2×150 bp.

Compute & Reproducibility: Containerized workflows (Snakemake/Nextflow), hardware-accelerated alignment, checksum-verified artifacts.

Illumina NovaSeq 6000

Illumina NextSeq 2000

Sample Requirements for eCLIP-Seq Assay

Item Accepted Types / Specs Recommended Amount / Range Key Notes
Sample Type Cell pellets, fresh-frozen tissues Cells or tissue sufficient for target IPs + controls Provide species/strain and culture/tissue details
Crosslinking UV 254 nm (intact cells/tissue) Fluence optimized per matrix No chemical crosslinkers or fixatives
Input Biomass Mammalian cell pellets 5–10×10⁶ cells per IP (typical) More may be needed for low-abundance RBPs
Tissues ≥50 mg per IP (typical) Homogenization method matters; consult us for fibrous tissues
Antibody Target-specific, IP-competent 5–10 µg per IP (validated) Provide WB/IP data or request antibody screening
Controls SMInput, optional IgG Match IP sample SMInput is used for normalization/statistics
Storage/Shipping Dry ice (–80 °C) N/A Use RNase-free tubes; include cold-chain log if available
Metadata Sample sheet N/A Antibody details, passage/treatment, expected isoforms
Exclusions Fixed samples (formalin, etc.) N/A Not compatible with eCLIP-Seq chemistry

Deliverables: What You Get from Our eCLIP-Seq Service

Raw & processed data: Demultiplexed FASTQ, aligned BAM, deduplicated BAMs, and QC summaries.

Peak & site files: BED/narrowPeak for peaks, crosslink site BED (truncation/mismatch evidence), IDR result tables.

Quantifications: Per-peak counts (unique molecules), SMInput-normalized enrichment, and differential binding tables with FDR.

Annotation layers: Feature overlaps (UTR/CDS/intron), splice-junction proximities, gene-level summaries.

Motif & enrichment outputs: Position weight matrices, motif logos (PNG/SVG), metagene plots, pathway/GO enrichment tables.

Genome browser tracks: Strand-specific bigWig coverage and crosslink signal tracks (UCSC/IGV-ready).

Reproducibility dossier: Replicate concordance, library complexity estimates, UMI duplicate rates, and pipeline parameter manifest.

Methods appendix: Detailed wet-lab and computational steps sufficient for Materials & Methods sections.

Genomic track with eCLIP signal highlighting a crosslink peak.

Peak Calling & Crosslink Site Tracks

eCLIP-Seq track showing RBP peak enrichment over input control.

Motif logo illustrating nucleotide preference at binding sites.

Motif Enrichment Logos

Sequence logo of enriched binding motif from eCLIP peaks.

Bar chart of binding site proportions in 5′UTR, CDS, 3′UTR, and introns.

Metagene / Feature Distribution Plot

Distribution of RBP binding sites across transcript features.

Scatter plot of replicate peak intensities with diagonal reproducibility.

Replicate Concordance / IDR Plot

Replicate comparison showing reproducible eCLIP binding peaks.

eCLIP-Seq vs Other RBP–RNA Mapping Methods: Which Should You Choose?

Category eCLIP-Seq RIP-Seq PAR-CLIP iCLIP / HITS-CLIP ChIP-Seq
Primary readout & resolution Near-base; crosslink-site evidence Enrichment only (low res) Single-nt (T→C) Base-level (truncations) DNA binding (not RNA)
Direct binding evidence Yes — UV crosslink, SMInput No (indirect possible) Yes — label + UV Yes — UV No
Crosslinking required Yes (UV 254 nm) No Yes (UV + labeling) Yes (UV) N/A (DNA)
Metabolic labeling No No Yes (e.g., 4SU) No No
Feasibility in primary tissue High High Often limited Moderate–High High (DNA only)
Controls / QC SMInput ± IgG; IDR reproducibility Input only Labeled input; assay QC Input; replicate QC Input; ChIP QC
Strengths High specificity; reproducible; widely adopted Simple; lower cost Precise crosslink sites Historical comparability Best for TF/chromatin
Limitations Needs validated antibodies; moderate input Indirect signals; high background Labeling not always feasible Workflow complexity Not an RNA method
Best use cases Transcriptome-wide, high-confidence RBP maps Broad targets when UV not possible Mechanistic mapping in model cells Studies needing legacy comparability Transcription/chromatin studies

You May Want to Know

Can you work with primary tissues or non-model organisms?

Yes—we support custom genome/annotation builds and matrix-specific prep to keep mapping accurate.

Can I use my own antibody?

Yes—send the datasheet and prior IP/WB evidence; we can also perform optional screening before full runs.

How many replicates should I run?

We co-design biological replicates based on hypothesis and expected variability so downstream statistics remain robust.

Do you accept analysis-only projects (FASTQ/BAM already generated)?

Yes—analysis-only is supported with a reproducible, version-pinned pipeline and a handover manifest.

Can you integrate eCLIP with our in-house RNA-seq or Ribo-seq?

Yes—provide sample mapping and we deliver coordinated analyses for expression, splicing, or translation context.

Will you prioritize sites for validation?

Yes—we rank candidates by enrichment, motif/context, feature location, and replicate consistency to guide follow-ups.

Can you handle engineered or tagged RBPs?

Yes—we discuss epitope/tag capture conditions and design appropriate controls for reliable pulldown.

Do you support custom genome versions/annotations?

Yes—reference builds and GTFs can be pinned to your requested versions for exact reproducibility.

How are multimappers and repetitive elements handled?

We apply reporting-aware alignment and targeted annotations so repetitive-region signals are interpretable.

Can I request single-nucleotide crosslink calls as well as peaks?

Yes—both site-level and peak-level outputs are available, delivered in standard browser-ready formats.

Can multiple RBPs or conditions be run in one study?

Yes—we design matched inputs and harmonized processing so contrasts remain comparable.

Is eCLIP-Seq suitable for primary tissue studies?

Yes—design and references are tailored so mapping remains reliable on primary material.

Do I need matched RNA-seq to interpret eCLIP results?

Not strictly, but matched RNA-seq or Ribo-seq strengthens functional interpretation.

Can eCLIP-Seq detect binding on lncRNAs and small RNAs?

Yes—noncoding targets are supported with ncRNA-focused annotations.

Is eCLIP-Seq better than RIP-seq for direct binding?

For direct, in-vivo contacts eCLIP is preferred; choose RIP when crosslinking isn't feasible.

Can I use a custom genome build or older GENCODE version?

Yes—pin the build/annotation you require for exact reproducibility.

Will you help prepare figures and a methods appendix?

Yes—figure-ready tracks/tables and a parameterized methods summary are included for easy downstream use.

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