Project description:Short-read sequencing of nascent RNA from exponentially growing S. pombe cells was employed to quantify co-transcriptional splicing and expression levels. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). Single-end Illumina sequencing data were generated from chromatin-associated, non-polyadenylated RNA (nascent RNA) and cytoplasmic mRNA for reference. In an experiment to test for splicing-associated gene expression changes cells were treated with 10mM Caffeine for 15minutes and nascent and mRNA-seq data were generated subsequently.
Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.
Project description:Long-read SMRT cDNA sequencing of nascent RNA from exponentially growing S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a transcriptome-wide PCR. SMRT DNA sequencing libraries were prepared subsequently.
Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently.
Project description:Evaluation of short-read-only, long-read-only, and hybrid assembly approaches on metagenomic samples demonstrating how they affect gene and protein prediction which is relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic, and metaproteomic data to evaluate the metagenomic-based protein predictions.
Project description:With an ability to compromise genome integrity, transposable elements (TEs) have significant associations with human diseases. Short-read sequencing has been used to study the expression of TEs; however, the highly repetitive nature of these elements makes multimapping a critical issue. Here we implement lasTEq, an improved quantification method by integrating long-read sequencing. Introducing computed transcript per million(TPM) counts from long-read sequencing as prior distribution during Expectation-Maximization(EM) model in short-read TE quantification, multi-mapped reads are re-assigned to correct expression values. Based on simulated short reads, lasTEq outperforms current quantitative approaches and is significantly favorable in capturing newly inserted TEs. We also verified that TEs quantified by lasTEq clearly related to euchromatins and heterochromatins in cell line samples. With lasTEq we anticipate that more accurate quantification can be performed, allowing novel functions of TEs to be uncovered.
Project description:Splicing and expression profiling of nascent and mRNA by RNA sequencing This SuperSeries is composed of the SubSeries listed below.