Transcriptome analysis by strand-specific sequencing of complementary DNA

Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Солдатов, А. В.

doi:10.1093/nar/gkp596

Cited by 769 publications

(633 citation statements)

References 21 publications

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“…1A). Polyadenylated RNA was purified from approximately 1000 embryos per time-point and converted into cDNA libraries for strand-specific, paired-end 76 bp sequencing on Illumina's HiSeq platform (see Methods; Parkhomchuk et al 2009;Levin et al 2010). On average, we obtained about 200-300 million reads per stage (more than two billion reads in total) (Supplemental Table 1).…”

Section: Assembly Of a High-confidence Embryonic Transcriptomementioning

confidence: 99%

“…The quality of the RNA and lack of contaminating ribosomal RNA were confirmed using the Agilent 2100 Bioanalyzer. Strand-specific libraries for 76-bp paired-end sequencing were prepared according to a modified UTP-method (Parkhomchuk et al 2009), as detailed by Levin et al (2010). Libraries were sequenced on the GA-analyzer (shield stage library) and on the Illumina HiSeq 2000 (all stages), at a depth of 200-300 million reads per library (for statistics on read counts, see Supplemental Table 1).…”

Section: Rna-seq Of Embryonic Time Coursementioning

confidence: 99%

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Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

Pauli¹,

Valen²,

Lin³

et al. 2011

Genome Res.

739

830

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Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.

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Section: Assembly Of a High-confidence Embryonic Transcriptomementioning

confidence: 99%

Section: Rna-seq Of Embryonic Time Coursementioning

confidence: 99%

Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

Pauli¹,

Valen²,

Lin³

et al. 2011

Genome Res.

739

830

View full text Add to dashboard Cite

show abstract

“…Many different types of noncoding transcripts have been reported to be associated with promoter regions or to be derived from sequences close to the transcription start site of genes (Davis and Ares, 2006;Parkhomchuk et al, 2009;Zhao et al, 2010). Theoretically, TUs for some of these transcripts may also appear in the intergenic regions.…”

Section: Design Of An Identifier System Of Arabidopsis Lincrnasmentioning

confidence: 99%

Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis

et al. 2012

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Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions of yeast and animal genomes play important roles in key biological processes. Yet, plant lincRNAs remain poorly characterized and how lincRNA biogenesis is regulated is unclear. Using a reproducibility-based bioinformatics strategy to analyze 200 Arabidopsis thaliana transcriptome data sets, we identified 13,230 intergenic transcripts of which 6480 can be classified as lincRNAs. Expression of 2708 lincRNAs was detected by RNA sequencing experiments. Transcriptome profiling by custom microarrays revealed that the majority of these lincRNAs are expressed at a level between those of mRNAs and precursors of miRNAs. A subset of lincRNA genes shows organ-specific expression, whereas others are responsive to biotic and/or abiotic stresses. Further analysis of transcriptome data in 11 mutants uncovered SERRATE, CAP BINDING PROTEIN20 (CBP20), and CBP80 as regulators of lincRNA expression and biogenesis. RT-PCR experiments confirmed these three proteins are also needed for splicing of a small group of introncontaining lincRNAs.

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“…Poly(A)-positive RNA was purified from 5 µg of total RNA (RNA integrity number ≥ 7.5) with the Dynabeads mRNA purification kit (Invitrogen), following the manufacturer's instructions. A strand-specific RNA-seq library was then prepared according to the deoxy-UTP approach as reported previously [54]. Amplified materials were loaded onto a flow cell and sequencing was carried out on the Illumina HiSeq2000 platform by running 90 cycles (paired-end design) according to the manufacturer's instructions.…”

Section: Library Preparation For a Strand-specific Poly(a)-positive Rmentioning

confidence: 99%

Hydrogen peroxide primes heart regeneration with a derepression mechanism

et al. 2014

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While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H 2 O 2 acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H 2 O 2 (~30 µM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H 2 O 2 formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H 2 O 2 by catalase overexpression markedly impaired cardiac regeneration while exogenous H 2 O 2 rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H 2 O 2 is an essential and sufficient signal in this process. Mechanistically, elevated H 2 O 2 destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H 2 O 2 plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H 2 O 2 potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.

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Transcriptome analysis by strand-specific sequencing of complementary DNA

Cited by 769 publications

References 21 publications

Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis

Hydrogen peroxide primes heart regeneration with a derepression mechanism

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