Gene Expression
Nebulas
Gene Expression NebulasSummary: We report the application of paired-end RNA sequencing for high throughput profiling of the Xenopus transcriptome in 23 distinct developmental stages. In total, we obtained over 900 million reads and the deep coverage allowed us to examine the transcriptome in detail. First, we found that ~150 genes are transcribed before embryonic genome activation when transcription is generally thought to be repressed. Second, we discovered thousands of novel splice junctions, the majority of which modify existing gene structures. Third, we curated a confident set of 6686 non-coding transcripts in 3859 genomic loci. Many of these non-coding RNAs are also developmentally regulated, which suggests that they may play important roles during embryogenesis. Finally, we found hundreds of contigs that cannot be aligned to the reference genome, which indicates that the current genome (XenTro3) is still unfinished. Our results will aid in the full assembly and annotation of the Xenopus tropicalis genome.
Overall Design: Examination of the transcriptome of Xenopus tropicalis from a 2-cell fertilized embryo to a stage 45 feeding tapole
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | polyA-containing RNAs were selected. |
| Library Construction Protocol: | The general Illumina mRNA-Seq library preparation workflow was followed with some modifications. First, polyA-containing RNAs were selected. The RNAs were then fragmented in 5X First-Strand Buffer (Invitrogen) at 85oC for 7-8 minutes. Random hexamers and SuperScript III (Invitrogen) were used to synthesize the first strand cDNA. Second strand cDNA synthesis was performed with dUTP in place of dTTP to mark the second strand. After end repair and addition of adenosine to the 3'ends, standard Illumina adapters were ligated. DNA fragments in the size range of 300 to 600bp were gel extracted and treated with UDG before each library was amplified using Phusion (Finnzymes). |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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