Transcriptome analysis by strand-specific sequencing of complementary DNA

By D. Parkhomchuk, T. Borodina, V. Amstislavskiy, Maria Banaru, Linda Hallén, Sylvia Krobitsch, Hans Lehrach, A. Soldatov. Nucleic Acids Research, 2009

950 citations recorded by the source corpus.

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/8bd68f6661cc19c11ea93968022c2d961e471fc4

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes (2012, methodology): “Strand-specific RNA-Seq libraries were prepared as described previously (Parkhomchuk et al., 2009) and sequenced on 1–3 lanes of Illumina’s HiSeq 2000 instrument.”
De novo transcript sequence reconstruction from RNA-Seq: reference generation and analysis with Trinity (2013, methodology): “Several methods are available for generating strand-specific RNA-seq 48,49 .”
RNA sequencing: advances, challenges and opportunities (2010, background): “Last, a third approach is the selective chemical marking of the second-strand cDNA synthesis products or RN”
Comprehensive comparative analysis of strand-specific RNA sequencing methods (2010, neutral): “We also compiled comparable data for two published libraries: a dUTP librar”
Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. (2012, methodology): “Identification of a stringent set of embryonic lncRNAs To identify mRNAs that exert their biological function as lncRNAs, we developed a highly stringent filtering pipeline aimed at re- Genome Research 579 www.genome.org moving transcripts with evidence for protein-coding potential (Methods; Fig.”
RNA Sequencing Data: Hitchhiker's Guide to Expression Analysis (2018, methodology): “140 cDNA and to degrade the dUTP labeled cDNA before PCR amplification [12] .”
RNA Sequencing and Analysis (2015, positive): “The preferred strategy to preserve strandedness is to incorporate a chemical label such as deoxy-UTP (dUTP) during synthesis of the second-strand cDNA that can be speci ﬁ cally removed by enzymatic digestion (Parkhomchuk et al. 2009).”
GC-Content Normalization for RNA-Seq Data (2011, methodology): “[26], as in [27] with the following modifications: fragmentation was carried out before cDNA synthesis as above and gel purification after PCR amplification was omitted.”