Transcriptome-Wide RBP Occupancy Studies
Uncover the full set of binding sites for an RNA-binding protein across the transcriptome to build comprehensive interaction maps.
PAR-CLIP Service — Single-Nucleotide Mapping of RNA–Protein Interactions
Pinpoint RBP binding sites with edit-anchored certainty. Creative Proteomics delivers end-to-end PAR-CLIP—from optimized 4SU/6SG labeling to CIMS-driven analytics—so you get defensible, single-nucleotide maps that explain splicing, stability, and translational control.
- Single-nucleotide proof: T→C/G→A diagnostics and CIMS for exact crosslink bases.
- Fit-for-purpose design: Labeling strategy, antibody/tag plan, and controls aligned to your model.
- Reproducible results: Replicate-aware statistics, IDR-style stability, and transparent QC.
- Publication-ready outputs: FASTQ, BED with edit counts, bigWig tracks, motif tables, and interpretive report.
- Comparative insights: Robust differential binding across treatments, knockouts, or time points.
What Is PAR-CLIP?
Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) captures native RNA–protein contacts inside living cells. Cells incorporate 4-thiouridine (4SU) or 6-thioguanosine (6SG) into nascent RNA. Long-wavelength UV-A crosslinking covalently locks RBPs to nearby RNA. After immunoprecipitation and sequencing, characteristic T→C (or G→A) conversions reveal crosslink sites at single-nucleotide precision. The result is a high-confidence binding map that resolves motifs, target regions, and condition-specific changes.
Typical Scientific Questions PAR-CLIP Can Answer
- Where does my RBP bind? Define exact contact sites and enriched transcript regions.
- Which motif drives binding? Discover sequence/structure motifs within crosslink peaks.
- Does binding change across conditions? Quantify differential binding under perturbations or time courses.
- How do binding events relate to function? Link sites to splicing, stability, localization, or translation control.
- Which RNAs comprise the regulated program? Prioritize targets and connect them to pathways and phenotypes.
Advantages of Our PAR-CLIP Service
Single-Nucleotide Resolution — T→C Diagnostic Editing
Identifies exact RBP contact sites by detecting crosslink-induced T→C transitions, enabling precise mapping beyond general enrichment zones.
Custom Labeling Strategy — 4SU or 6SG Optimization
Supports both 4-thiouridine and 6-thioguanosine labeling, with tailored dose and incubation guidance to maximize incorporation efficiency and minimize cytotoxicity.
Controlled UV-A Crosslinking — 365 nm Irradiation
Applies temperature-monitored UV-A exposure to preserve cell viability while ensuring efficient covalent bond formation between RNA and protein.
Validated IP Workflow — High-Specificity Enrichment
Combines epitope-tagged or antibody-based immunoprecipitation with rigorous control conditions (no-label, IgG) to reduce background and increase peak confidence.
Bias-Minimized Library Prep — Adapter & Cycle Titration
Employs ligation-based construction with adapter sequence optimization and cycle titration to limit amplification bias and preserve true site diversity.
CIMS-Enabled Analysis — Crosslink-Induced Mutation Site Detection
Pinpoints crosslink sites with nucleotide-level accuracy using computational detection of CIMS, enhancing resolution and reproducibility.
Replicate-Aware Statistics — Robust Differential Binding
Incorporates biological replicates into statistical models for differential binding, ensuring stable peak calling across sample groups with IDR-style confidence.
Molecular Interaction Analysis
Service Scope
Workflow and Instrumentation
Sample Requirement
Deliverables
Which to Choose
FAQ
Get a Custom Proposal
Scope of PAR-CLIP Services at Creative Proteomics
Condition-Dependent Binding Landscape Projects
Compare RBP occupancy under perturbations such as compound treatment, environmental stress, or gene knockout to identify context-specific regulation.
Non-Coding RNA Interactome Profiling
Map protein contacts on lncRNAs, snoRNAs, or viral RNAs to clarify their functional roles and regulatory partners.
UTR- and Splicing-Focused Investigations
Zoom in on 5'/3' UTRs or intron–exon boundaries to reveal how RBPs control transcript stability, translation, and isoform selection.
Motif and Sequence Preference Characterization
Determine sequence and structure elements that drive RBP recognition, enabling predictive modeling of binding specificity.
Comparative RBP Target Studies
Profile multiple RBPs in parallel to highlight competitive, redundant, or cooperative regulatory relationships.
Custom Targeted RBP Projects
Support projects using client-supplied antibodies or engineered tags (FLAG, HA, GFP) when specialized enrichment is required.
Our PAR-CLIP Service Workflow
1
Study Design & Acceptance Criteria
Define model, labeling nucleoside, capture strategy, controls, and statistical power.
2
Metabolic Labeling & UV-A Crosslinking
Optimize incorporation; perform temperature-controlled crosslinking; verify viability proxies and labeling success.
3
Immunoprecipitation & Controlled Fragmentation
Enrich RBP–RNA complexes; tune RNase to target insert length; include IgG and no-label controls.
4
Library Construction & Sequencing
Adapter ligation, reverse transcription, and careful PCR cycle selection; verify library size/molarity; sequence.
5
Computational Analysis & Interpretation
Trim and map with T→C awareness; call peaks and CIMS; derive motifs; assess replicate stability; run differential binding; deliver browser-ready tracks and prioritized targets with context.
PAR-CLIP Instrumentation & Technical Capabilities
Labeling: 4SU or 6SG metabolic incorporation with dose and schedule optimization.
Crosslinking: UV-A systems with monitored irradiance and thermal control to protect cellular integrity.
QC instrumentation: Bioanalyzer/TapeStation for fragment/library sizing; HPLC/UV or proxy assays for labeling checks; immuno-validation for IP success.
Sequencing platforms: Illumina short-read systems suitable for high-complexity libraries.
Bioinformatics stack: T→C-aware mappers, peak callers with CIMS, motif discovery, metagene and region enrichment, replicate concordance (e.g., IDR-style checks), and pathway analysis.
Data formats: FASTQ, BED peaks with T→C counts, bigWig coverage tracks, count matrices, annotated target tables, and an interpretive PDF/HTML report.
Sample Requirements for PAR-CLIP Assay
| Item | Preferred Specification | Notes / QC on Receipt |
| Biological material | Viable cultured cells (adherent or suspension), primary cells, or organoids | PAR-CLIP requires live cells for metabolic labeling; fixed tissues are not suitable |
| Minimum input per IP | Typically 5–20 × 106 cells per IP (RBP- and antibody-dependent) | Final input is set during study design based on RBP abundance and IP efficiency |
| Replicates | ≥3 biological replicates per condition (recommended) | Enables robust differential binding and reproducibility checks |
| Labeling nucleoside | 4-thiouridine (4SU) or 6-thioguanosine (6SG) | Choice guided by cell tolerance and analysis goals; labeling efficiency will be verified |
| Antibody / tag | Validated antibody or epitope tag (e.g., FLAG/HA/GFP) | Provide datasheet and prior evidence; co-sourcing/validation can be arranged |
| Controls | No-label control and IgG IP control | Required for background estimation and confident peak calling |
| Culture conditions | Provide base medium, supplements, and any additives | Maintain consistent passage number and confluency; record lot info if applicable |
| Sample format | Cell pellets on dry ice or live shipment for on-site labeling (by prior arrangement) | Clearly labeled tubes with condition/replicate IDs; avoid freeze–thaw cycles |
| Shipping | Cold-chain (dry ice) with tamper-resistant secondary containment | Include packing list and emergency contact; follow local and IATA guidelines |
| Metadata | Cell line/primary source, passage, growth conditions, antibody details, planned contrasts | Complete metadata accelerates transparent methods and reproducible analysis |
Deliverables: What You Get from Our PAR-CLIP Service
- Prioritized target list with binding site coordinates and confidence tiers.
- Motif logos and sequence context aligned to reference transcript features.
- Region-level enrichment (5' UTR, CDS, 3' UTR, introns, lncRNAs).
- Differential binding tables with effect sizes and false discovery control.
- Pathway overlays linking RBP targets to biological programs.
- Actionable commentary mapping findings to your hypotheses and next experiments.
T→C Mutation Signature Plot
T→C transition frequency aligned to PAR-CLIP peak center, showing a sharp summit at 0 nt versus a flat control background—evidence of single-nucleotide crosslinking specificity.
CIMS Peak Track
Genome browser view with PAR-CLIP signal (bigWig), called peak region, and red CIMS lollipops concentrated near the summit; gene model on the negative strand for positional context.
Motif Discovery & Sequence Logo
Sequence logo from enriched PAR-CLIP peaks, highlighting a U/A-biased 10-nt motif; example E-value indicates strong enrichment.
Differential Binding Volcano Plot
Volcano plot comparing conditions (e.g., WT vs KO): significant targets with |log2FC| ≥ 1 and FDR ≤ 0.05 highlighted as increased (▲) or decreased (▼) binding; top hits labeled.
PAR-CLIP vs. eCLIP/iCLIP vs. RIP-seq— Pick the Right Method for Your Study
| Method | PAR-CLIP | eCLIP/iCLIP | RIP-seq |
| Crosslink principle | UV-A (365 nm) after 4SU/6SG metabolic labeling | UV-C (254 nm) direct crosslink | Native or light crosslink; co-IP of RNA with RBP |
| Diagnostic signal | T→C (or G→A) edits; CIMS pinpoint base | Truncations/overhangs; crosslink-derived stops | None (enrichment only) |
| Resolution | Single-nucleotide (edit-anchored) | Near-nucleotide (site inferred) | Transcript-level (co-precipitated lists) |
| Labeling required | Yes (4SU/6SG) | No | No |
| Suitable material | Label-tolerant live cells, organoids | Cell lines, primary cells, tissues (with UV) | Broad (cells/tissues/lysates) |
| Input need (per IP) | Moderate; varies with RBP abundance | Moderate; efficient with good antibodies | Low–moderate |
| Controls | No-label + IgG controls recommended | Size-matched input + IgG controls | IgG and input controls essential |
| Library features | Small-RNA–style ligation; edit-aware | Ligation with size-matched inputs; truncation capture | Standard RNA IP libraries |
| Primary outputs | BED peaks with T→C/G→A counts, CIMS, bigWig tracks | BED peaks (crosslink signatures), bigWig tracks | Target RNA lists/enrichment tables |
| Motif discovery power | High (edit-anchored peak cores) | High with stringent peaks | Limited/ambiguous |
| Differential binding robustness | High (edit signal stabilizes peaks) | High with replicates/IDR | Moderate; higher background |
| Background/false-positive risk | Low (diagnostic edits) | Low–moderate | Highest (co-IP carryover) |
| When it excels | Mechanism studies needing exact sites; mutation/compound effects | When labeling not feasible but site maps are needed | Quick screens, low input, early discovery triage |
| Key limitations | Requires metabolic labeling; not ideal for fixed tissue | No edit signature; UV-C can reduce RNA yield | No base-level sites; interpretation can be noisy |
| QC focus | Label uptake, crosslink yield, T→C fraction, duplicate rate | Crosslink efficiency, size-matched input, reproducibility | IP specificity, rRNA carryover, enrichment consistency |
| Bioinformatics complexity | Edit-aware mapping & CIMS; motif/region analysis | Truncation-aware mapping; IDR/replicate stability | Enrichment stats; limited site modeling |
If your model supports 4SU/6SG labeling and you need base-level, mechanistic certainty, choose PAR-CLIP. If labeling isn't viable, eCLIP/iCLIP provides strong site maps. Use RIP-seq for fast, low-barrier screening when precision is not critical.
You May Want to Know
Can PAR-CLIP work if my cells label poorly with 4SU/6SG?
We run a brief feasibility test (dose–response and viability proxy). If uptake is marginal, we adjust dosing schedules, switch nucleoside (4SU↔6SG), or recommend an eCLIP path for the same model.
What if my antibody is weak or shows off-target bands?
We can evaluate orthogonal capture (epitope tags like FLAG/HA/GFP) or use antibody pre-clearing and harsher washes. We also assess enrichment by IP-qPCR or Western prior to full runs.
Do I need biological replicates for PAR-CLIP?
Yes for inference (e.g., condition contrasts). Two replicates are minimal; three provide robust stability checks (IDR-style) and more reliable motif enrichment.
How do you decide sequencing depth?
Depth is proportional to RBP abundance, transcriptome complexity, and planned contrasts. We scale reads to achieve stable peak discovery and motif recovery without over-sequencing.
How are multi-mapping reads handled?
We use edit-aware alignment with conservative placement rules. Ambiguous loci are flagged or excluded in high-stringency sets and retained (labeled) in exploratory sets.
Can you analyze non-coding RNAs, viral RNAs, or repetitive regions?
Yes—outputs include flagged context for repeats and non-coding features; interpretation notes explain confidence limits and alternative mappings.
What controls are recommended beyond IgG/no-label?
Size-matched input (SMI) or mock IP can be added. For perturbation studies, spike-in or anchored normalization improves cross-sample comparability.
How do you prevent RNase over-digestion during fragmentation?
We titrate RNase on a pilot lysate and verify insert sizes by electropherogram before committing; settings are locked into the run sheet.
How do you distinguish true edits from sequencing errors or SNPs?
We require edit enrichment over controls, evaluate local base-error models, consult known SNP panels when provided, and apply position-wise filters around peak cores.
Can PAR-CLIP data be integrated with RNA-seq or PRO-seq?
Yes. We can correlate binding with expression or transcriptional flux, highlight gain/loss sites with matched differential analyses, and provide pathway overlays.
What determines whether I should use 4SU or 6SG?
Cell tolerance and downstream goals. 4SU yields T→C diagnostics; 6SG yields G→A. We pick the nucleoside with the best incorporation–viability balance for your model.
How stable are motif calls across replicates or conditions?
We compute motif stability using resampling across peak subsets and report consensus logos with enrichment statistics and positional preference (e.g., UTR vs CDS).
How are background peaks filtered?
Thresholds combine edit fraction, replicate concordance, control subtraction, and local sequence context. We report both “stringent” and “relaxed” peak sets.
Can you support multi-RBP projects under the same conditions?
Yes. We coordinate shared controls and normalization so overlaps/competition are comparable across RBPs.
What happens if labeling affects cell phenotype?
We can shorten labeling exposure, reduce dose, or test the alternative nucleoside. If biology is sensitive, we propose eCLIP as a non-label substitute.
Do you report site confidence levels?
Yes—peaks are tiered by edit density, replicate support, and control contrast. Each tier includes guidance for downstream validation (e.g., mutagenesis, reporter assays).
How do you handle batch effects across runs?
We stabilize with shared controls, aligned processing, and post-hoc normalization. Batch diagnostics are included in the QC dashboard.
Can you localize binding relative to transcript features?
We provide metagene profiles and feature enrichment (5'UTR, CDS, 3'UTR, intron, lncRNA), with separate summaries for primary and alternative isoforms when annotations allow.
What formats are easiest for my team to reuse?
Browser tracks (bigWig), coordinate files (BED with edit counts), and tidy target tables. We can include simple notebooks or scripts for quick re-plots if requested.
