PAS Sequencing

Poly(A) Site Sequencing (PAS-Seq) is a next-generation sequencing-based method that is extensively used in quantitatively profiling of RNA polyadenylation at the transcriptome level. PAS Sequencing provides valuable information for identifying both poly(A) junctions in mRNAs and noncoding RNAs in an accurate and comprehensive manner, as well as provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq studies of different bio-development processes and diseases provide us with a systematic approach to understand biological regulation beyond the level of gene expression, which is a research direction worth to be further studied.

Overview

The alternative polyadenylation (APA) mechanism generates a distinct 3’-termini on mRNAs and other transcripts. 3’-UTR cis-regulatory elements can affect the regulation of the transcription. The approach of studying APA allows researchers to gain more understanding of miRNA regulation, mRNA stability and localization, and mRNA translation. In addition to 3’-Tag-RNA-Seq method, PAS-Seq provides an alternative method for APA analysis. In this process, mRNA is cleaved into fragments of 150 bp or so. Template replacement is then performed using primers with oligo-dT to generate cDNA for subsequent sequencing.

Features

High Performance Transcriptome-Wide Bioinformatics Analysis One-stop Service
High resolution, sensitivity, and specificity A systematic approach to understand biological regulation beyond the level of gene expression 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.

PAS Sequencing

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Biostatistical analysis - transcript copy number comparisons, expression analysis, length distribution, multi-parameter data analysis, etc.
  • GO and KEGG enrichment analysis
  • Detect novel and rare transcripts
  • Target gene prediction and functional analysis
  • Alternative polyadenylation
  • UTR isoform 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. Ashar-Patel A, Kaymaz Y, Rajakumar A, et al. FLT1 and transcriptome-wide polyadenylation site (PAS) analysis in preeclampsia. Scientific reports. 2017, 7(1): 1-14.
  2. Yao C, Shi Y. Global and quantitative profiling of polyadenylated RNAs using PAS-seq. Polyadenylation: Springer; 2014. p. 179-85.
  3. Shepard PJ, Choi E-A, Lu J, et al. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA. 2011, 17(4): 761-72.
* For Research Use Only. Not for use in diagnostic procedures.


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