RNA Immunoprecipitation Sequencing

RNA immunoprecipitation sequencing (RIP-seq) is a next-generation sequencing (NGS)-based method to comprehensively study the situation of intracellular RNA and its binding protein, a dynamic process of the post-transcriptional regulatory network. We provide a one-stop RIP-seq service to help customers discover new RNA-protein interaction sites and explore more gene regulation functions.

Overview

Currently, except for a few RNAs that can function alone in the form of ribozymes, most RNAs are combined with proteins to form RNA-protein complexes. RNA-binding proteins (RBPs) play an important role in the process of gene regulation such as RNA synthesis, alternative splicing, modification, transport, and translation. Therefore, studying the interaction of RNA and protein is the key to exploring RNA functions. RNA immunoprecipitation (RIP) is an important technique for studying the interaction of protein and RNA in vivo. The technology uses antibodies against the target protein to precipitate the corresponding RNA-protein complex, and analyze RNA after separation and purification. The advent of sequencing technologies, coupled to various RIP chemistries, has enabled the simultaneous detection of thousands of bound transcripts in a single experiment. Based on RIP, RIP-seq uses specific antibodies to immunoprecipitate RNA-binding proteins or specially modified RNAs. After RNA isolation, Illumina sequencing is used to study RNA regions or types specifically bound by specific proteins across the full transcriptome, and differences between multiple samples can be compared. Our company provides RIP-seq to obtain insights into not just the well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs. RIP-Seq is a powerful tool for understanding the dynamic process of post-transcriptional regulatory networks. It provides researchers with the possibility to better understand the mechanisms of post-transcriptional regulation and even gene expression in vivo.

Features

High Coverage High Resolution Strict Quality Control One-stop Service
The method can screen and identify protein binding sites in whole genome. Combined with high-throughput sequencing technology, new protein binding sites can be found. Monitor experiment quality throughout the process to ensure customers get high-quality data. 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;
strand-specific cDNA libraries.

Sequencing

3. Sequencing

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

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

RNA Immunoprecipitation Sequencing

In-depth data analysis:

  • Raw data quality control
  • Quality assessment of sequencing data
  • Peak calling and visualization
  • Peak identification
  • Peak distribution
  • Peaks annotation
  • Motif search of enrichment sites
  • Differential binding analysis
  • Alterative splicing analysis
  • GO and KEGG pathway analysis
  • Clustering analysis and enrichment analysis

Sample Requirements

RNA sample quantity ≥ 50 ug.
OD260/280 ≥ 1.8, OD260/230 ≥ 1.5.
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. Wessels H H, Hirsekorn A, Ohler U, et al. Identifying RBP Targets with RIP-seq. Methods Mol Biol, 2016, 1358:141-152.
  2. Zeng Y, Wang S, Gao S, et al. Refined RIP-seq protocol for epitranscriptome analysis with low input materials. PLoS Biol, 2018, 16(9): e2006092.
  3. Mugo E, Erben E D. Identifying Trypanosome Protein-RNA Interactions Using RIP-Seq. Methods Mol Biol, 2020, 2116: 285-294.
  4. Nicholson C O, Friedersdorf M B, Bisogno L S, et al. DO-RIP-seq to quantify RNA binding sites transcriptome-wide. Methods, 2017, 118: 16-23.
* For Research Use Only. Not for use in diagnostic procedures.


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