Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of yeast using samples prepared with actinomycin D to exclude antisense artifacts

ABSTRACT: Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Zhenyu Xu

PROVIDER: E-TABM-361 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D.

Perocchi Fabiana F Xu Zhenyu Z Clauder-Münster Sandra S Steinmetz Lars M LM

Nucleic acids research 20070926 19

Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthe ...[more]

PMID: 17897965

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Project description:Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or a few conditions. We applied direct high-throughput sequencing of cDNAs, complemented with different expression data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including exponential proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at unprecedented resolution. Unlike hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not actively transcribed or rapidly degraded. The combined sequencing and strand-specific array data provided rich information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus refining the existing genome annotation. Sequence trans-reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes. Splicing efficiency was largely coordinated with transcriptional efficiency, and hundreds of introns showed regulated splicing as a function of cellular proliferation or differentiation.

Dataset Information

Transcription profiling of yeast using samples prepared with actinomycin D to exclude antisense artifacts

Publications

Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D.

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