GoldCLIP Sequencing

Compared to traditional CLIP-Seq (crosslinking and immunoprecipitation-Seq), which is technically challenging and requires radioactive labeling and PAGE purification for RNA library recovery, GoldCLIP-Seq (Gel-omitted and ligation-dependent CLIP-Seq) omits all gel purification steps and offers a more convenient and faster approach to study protein-RNA interactions, as well as the functional mechanism of lncRNA/circRNA and identification of miRNA targets. This non-isotopic method allows for highly reproducible CLIP-Seq that is compatible with diverse crosslinking conditions.

Overview

The GoldCLIP assay generally falls into two main steps: the construction of HaloTag fusion protein plasmids, and the expression and detection of the HaloTag fusion protein. The atomic mass of HaloTag is 33kDa and it can bind to targeted protein either on the N-terminal or C-terminal. When HaloTag is inserted into the targeted protein, the protein-RNA complex with HaloTag is covalently coupled to magnetic beads containing halogen ligands and can be separated. After adding a ligand to the 3'-end of RNA, protein denaturing agents such as Urea, Guanidine, and SDS can be used to carry out in vitro purification. Protein is degraded and targeted RNA can be recycled for cDNA library preparation, sequencing, and data analysis. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

Features

High Sensitivity High Coverage Wide Application One-stop Service
Each sample can get millions of sequence tags and find rare protein binding sites on the transcriptome. Screen and identify protein binding sites across entire transcriptome. Suitable for research on splicing factor RNA binding profiles, miRNA targets, etc. Provides one-stop service for library construction, sequencing, sample QC and data analysis.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

RNA fragmentation; cDNA library preparation.

Sequencing

3. Sequencing

Illumina HiSe; PE 50/75/100/150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

GoldCLIP Sequencing

In-depth data analysis:

  • Sequencing quality distribution
  • Peak calling and visualization
  • Peaks width and distance analysis
  • Identify potential miRNA target(s)
  • Distribution analysis of peak.
  • Analyze microRNA-mRNA interactions
  • Identification of protein binding site
  • Differential binding analysis
  • Motif search of enrichment sites
  • GO and KEGG pathway analysis

Sample Requirements

RNA sample quantity ≥ 50 ug.

Please make sure that the RNA is not significantly degraded.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

  1. Gu J, Wang M, et al. GoldCLIP: gel-omitted ligation-dependent CLIP. Genomics, proteomics & bioinformatics. 2018, 16(2): 136-43.
* For Research Use Only. Not for use in diagnostic procedures.


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