SEQanswers

Go Back   SEQanswers > Literature Watch



Similar Threads
Thread Thread Starter Forum Replies Last Post
Strand specific RNA-seq data from dUTP protocol ZhengXia Bioinformatics 8 10-16-2014 05:36 AM
RNA-Seq: High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Newsbot! Literature Watch 1 01-22-2013 12:48 AM
RNA-Seq: Method for improved Illumina sequencing library preparation using NuGEN Ovat Newsbot! Literature Watch 0 04-14-2011 03:50 AM
RNA prep for Multiplexed- Strand Specific RNA seq cmjluckey Sample Prep / Library Generation 0 02-21-2011 08:25 AM
RNA-Seq: Comprehensive comparative analysis of strand-specific RNA sequencing methods Newsbot! Literature Watch 0 08-17-2010 03:00 AM

Reply
 
Thread Tools
Old 09-29-2011, 07:00 AM   #1
Newsbot!
RSS Posting Maniac
 

Join Date: Feb 2008
Posts: 1,438
Default RNA-Seq: A Strand-Specific Library Preparation Protocol for RNA Sequencing.

Syndicated from PubMed RSS Feeds

A Strand-Specific Library Preparation Protocol for RNA Sequencing.

Methods Enzymol. 2011;500C:79-98

Authors: Borodina T, Adjaye J, Sultan M

Abstract
The analysis of transcriptome, which was over the past decade based mostly on microarray technologies, is now being superseded by so-called next generation sequencing (NGS) systems that changed the way to explore entire transcriptome. RNA sequencing (RNA-Seq), one application of NGS, is a powerful tool, providing information not only about the expression level of genes but also further about the structure of transcripts as it enables to unequivocally identify splicing events, RNA editing products, and mutations in expressed coding sequences within a single experiment. Herein, we describe step by step the deoxy-UTP (dUTP) strand-marking protocol [Parkhomchuk, D., Borodina, T., Amstislavskiy, V., Banaru, M., Hallen, L., Krobitsch, S., Lehrach, H., Soldatov, A. (2009). Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res.37(18), e123], which has been recently reviewed as the leading protocol for strand-specific RNA-Seq library preparation [Levin, J. Z., Yassour, M., Adiconis, X., Nusbaum, C., Thompson, D. A., Friedman, N., Gnirke, A., Regev, A. (2009). Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods7(9), 709-715]. The procedure starts with the isolation of the polyA fraction (mRNA) within a pool of total RNA, followed by its fragmentation. Then double-stranded (ds) cDNA synthesis is performed with the incorporation of dUTP in the second strand. The ds cDNA fragments are further processed following a standard sequencing library preparation scheme tailored for the Illumina sequencing platform: end polishing, A-tailing, adapter ligation, and size selection. Prior to final amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase (UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing.


PMID: 21943893 [PubMed - as supplied by publisher]



More...
Newsbot! is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 12:12 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO