RNA-Seq: High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation.

Newsbot! · 08-03-2011, 03:20 AM

Syndicated from PubMed RSS Feeds

High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation.

Cold Spring Harb Protoc. 2011;2011(8)

Authors: Zhong S, Joung JG, Zheng Y, Chen YR, Liu B, Shao Y, Xiang JZ, Fei Z, Giovannoni JJ

INTRODUCTION Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome assembly, and accurate digital gene expression analysis. This protocol describes a simple and robust method to generate ssRNA-Seq libraries for the Illumina sequencing platform. It has significantly increased the throughput to 96 libraries in a two-day preparation while simultaneously lowering the reagent costs to below ten dollars per library. It is compatible with both single-read and paired-end multiplex sequencing and, most importantly, its data can also be used with existing conventional RNA-Seq data. This is a significant advantage, because it enables researchers to switch to ssRNA-Seq even if a large amount of data has already been generated by the nonstrand specific methods.

PMID: 21807852 [PubMed - as supplied by publisher]

More...

augenetech · 01-21-2013, 11:48 PM

I run through the CSH strand-specific RNA sequencing protocol. There is a purification step using RNAcleanXP after the first strand synthesis. But then only use the purified first strand product, buffer, dNTPs, RNase and DNA polymerase I for second-strand synthesis. I wounder if any primers needed?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
HiSeq multiplexing for strand specific RNA library prep	ngon	Sample Prep / Library Generation	2	10-18-2011 08:32 AM
RNA-Seq: A Strand-Specific Library Preparation Protocol for RNA Sequencing.	Newsbot!	Literature Watch	0	09-29-2011 06:00 AM
RNA-Seq: Method for improved Illumina sequencing library preparation using NuGEN Ovat	Newsbot!	Literature Watch	0	04-14-2011 02:50 AM
RNA prep for Multiplexed- Strand Specific RNA seq	cmjluckey	Sample Prep / Library Generation	0	02-21-2011 07:25 AM
RNA-Seq: Comprehensive comparative analysis of strand-specific RNA sequencing methods	Newsbot!	Literature Watch	0	08-17-2010 02:00 AM

08-03-2011, 03:20 AM	#1
Newsbot! RSS Posting Maniac Join Date: Feb 2008 Posts: 1,443	RNA-Seq: High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Syndicated from PubMed RSS Feeds High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Cold Spring Harb Protoc. 2011;2011(8) Authors: Zhong S, Joung JG, Zheng Y, Chen YR, Liu B, Shao Y, Xiang JZ, Fei Z, Giovannoni JJ INTRODUCTION Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome assembly, and accurate digital gene expression analysis. This protocol describes a simple and robust method to generate ssRNA-Seq libraries for the Illumina sequencing platform. It has significantly increased the throughput to 96 libraries in a two-day preparation while simultaneously lowering the reagent costs to below ten dollars per library. It is compatible with both single-read and paired-end multiplex sequencing and, most importantly, its data can also be used with existing conventional RNA-Seq data. This is a significant advantage, because it enables researchers to switch to ssRNA-Seq even if a large amount of data has already been generated by the nonstrand specific methods. PMID: 21807852 [PubMed - as supplied by publisher] More...

01-21-2013, 11:48 PM	#2
augenetech Junior Member Location: hk Join Date: May 2010 Posts: 1	I run through the CSH strand-specific RNA sequencing protocol. There is a purification step using RNAcleanXP after the first strand synthesis. But then only use the purified first strand product, buffer, dNTPs, RNase and DNA polymerase I for second-strand synthesis. I wounder if any primers needed?