CAGE Sequencing

CAGE- Seq (Cap Analysis Gene Expression and deep Sequencing) is a technique that integrates the identification of capped RNAs and high-throughput sequencing, which can identify the Transcription Start Sites (TSSs) of the entire intracellular mRNA. CAGE has been widely used in the accurate profiling of promoters and 5'-UTR, providing extensive information on transcription factor binding motif, novel biomarkers, transcriptomic heterogeneity, etc.

Overview

The vast majority of genes have two or more TSSs where different TSSs lead to varied regulatory effects by upstream 5’-untranslated region (5'-UTR). While different 5'-UTR may contain distinct acting elements, and different start sites lead to completely different signals in response to gene expression. Thus, one gene may be regulated by different promoters, resulting in different expressions, which may lead to the occurrence of certain diseases. CAGE-Seq occurs to offer powerful solutions to analyze TSSs by identifying RNA cap sites. In CAGE-Seq assay, the mRNA sequences are fragmented and dephosphorylated, and the end phosphoric groups without cap protection are removed by phosphatases. Then the caps are removed, leaving only an unprotected 5' -terminal sequence with no exposed hydroxyl groups. Sequencing requires two joints, one of which is designed to link the hydroxyl exposed phosphoric acids, and the other to link the normal sequences, which makes the sequences that are formerly protected by caps can be sequenced. Finally, the CAGE sequence data is aligned to the reference genome, and the transcription start sites and the corresponding 5'-UTR information can be obtained, providing an effective alternative method for genome-wide transcriptional profiling.

Features

High Throughput High Accuracy Bioinformatics Analysis One-stop Service
Cost-effective transcriptional profiling solution for large sample-size assays. Accurate mapping of TSSs with single-nucleotide resolution. 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 HiSeq;
PE 50/75/100/150.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

CAGE Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Data quality control;
  • Reference-based mapping;
  • SNP calling;
  • Gene annotation;
  • Promoter site analysis;
  • Motif finding for transcription factor binding sites;
  • Identification of bidirectional enhancer RNA;
  • Copy number analysis.

Sample Requirements

RNA sample (concentration ≥ 1 ng/µL, quantity ≥ 5 µg)
1.8 ≤ OD260/280 ≤ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0.
Please make sure that the RNA is not degraded nor contaminated.

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, full statistical analysis & alignments, custom analysis reports on customer request.

References:

  1. Adiconis X, Haber AL, Simmons SK, et al. Comprehensive comparative analysis of 5'-end RNA-sequencing methods. Nature methods. 2018, 15: 505-11.
  2. Kawaji H, Lizio M, Itoh M, et al. Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing. Genome Res. 2014, 24(4): 708-17.
* For Research Use Only. Not for use in diagnostic procedures.


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