RAP Sequencing

RNA Antisense Purification (RAP) is an RNA-centric biochemical purification method that is used to study the interaction between RNA and DNA, RNA and proteins. Integrating RAP with next-generation sequencing (NGS)-based RNA-Sequencing, RAP-Seq is a general approach for mapping the localization of a given lncRNA across the genome as well as studying their functions and mechanisms.

Overview

RAP is the critical step for library preparation of RAP-Seq, which involves the isolation of lncRNAs with a set of long antisense oligos that are designed and used to capture the probe-RNA-chromatin complexes. In this process, cells are firstly crosslinked and lysed, followed by DNase I chromatin digestion of DNA fragments. The RNA mixture is dephosphorylated and the antisense probes are hybridized with lncRNAs. And the RNA probes are pulled-down with streptavidin to separate the cDNA fragments of interest. After the captured complexes are eluted, a second adaptor is ligated and amplified before sequencing. RAP-Seq requires minimal amplification steps during RNA sequencing post-purification of lncRNA complex and can be used in the genomic mapping of lncRNA targets,

Features

High Sensitivity High Specificity Bioinformatics Analysis One-stop Service
High resolution and sensitivity Long RNA probe length provides high binding affinity to the target lncRNA Our integrated bioinformatics pipeline can be tailored to suit your project. 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

RAP Sequencing

In-depth data analysis:

  • Profiling of lncRNA
  • Predict novel lncRNA
  • Detect novel and rare transcript variants
  • Differential expression analysis of transcripts
  • Target gene prediction and functional analysis
  • GO/KEGG analysis
  • Annotation

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. Engreitz J, Lander ES, Guttman M. RNA antisense purification (RAP) for mapping RNA interactions with chromatin. Nuclear Bodies and Noncoding RNAs; 2015. p. 183-97.
  2. Engreitz J. RNA Antisense Purification (RAP): Experimental Protocols. 2014.
* For Research Use Only. Not for use in diagnostic procedures.


Inquiry

  • Verification code
logo
Copyright © 2021 CD Genomics. All rights reserved.