High-throughput RNA sequencing analysis of WT, rdm16-2 and sta1 mutants
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE44635 | GEO | 2013/02/26
SECONDARY ACCESSION(S): PRJNA190679
REPOSITORIES: GEO
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Project description:Using Illumina paired-end RNA-seq technology to examine the genome-wide gene expression and intron-retention events in WT, rdm16-2 and sta1 RNA-seq analysis of 4-week-old leaves of WT, rdm16-2 and sta1 mutants
2013-02-26 | E-GEOD-44635 | biostudies-arrayexpress
Project description:The maize Rough endosperm3 (Rgh3) gene encodes an ortholog of the human essential splicing factor, ZRSR2. To test whether a mutation in Rgh3 affects mRNA splicing, we compared rgh3 mutants and wild-type sibling transcriptomes in an RNA-seq experiment. Twelve libraries were constructed with mRNA extracted from the roots and shoots of three seedlings of each genotype. The libraries were multiplexed and sequenced on one lane of the HiSeq 2000 platform. The run produced 149 million paired-end 100 bp reads that mapped to 35,028 genes. Two approaches were used to analyze the dataset. In the first approach, Mosaik2, FreeBayes, GSNAP, and Cufflinks were used to identify differences in transcript isoform abundance in a SNP-tolerant fashion. During reverse-transcription PCR validation, six examples of intron retention were found to occur more frequently in rgh3 seedlings, and all six introns were members of a rare class of introns called U12-type introns. The second approach utilized a t-test to determine whether more reads were mapped to U12-type introns in rgh3 libraries relative to wild-type libraries. Out of all U12-type introns within genes that are expressed at a early seedling stage, 43% exhibit splicing defects in rgh3 mutants. These U12-type intron splicing defects include intron retention and cryptic splice site activation. We report that the rgh3 mutation specifically impairs the U12-type intron splicing.
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Project description:Targeted paired-end sequencing of cDNA from unfragmented nascent RNA from exponentially growing S. cerevisiae cells was employed to obtain Pol II transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010) or enriched from total RNA with polyadenylated RNA depletion. The nascent 3â end was labeled with a 3â DNA adaptor through ligation. A PCR with a forward primer in the first exon of select intron-containing genes amplifies nascent transcripts of specific genes and ensures sequencing adaptor attachment for paired-end sequencing. With this approach co-transcriptional splicing progression with distance from the intron end could be analyzed for 87 genes. Note that the unmapped and mapped data also include genes that did not pass the read coverage requirements in SMIT analysis. Nascent RNA profiles for mainly intron-containing genes were generated with paired-end sequencing with Illumina HiSeq technology.
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Project description:Abstract: Paired-end sequence data has been generated using polyA selected RNA from a range of zebrafish tissues and developmental stages using the Illumina Genome Analyzer. These data have been used to improve the gene annotation of the zebrafish genome. Study description: Zebrafish total RNA was extracted from embryonic and adult tissue, then polyA selected. After fragmentation and reverse transcription Illumina sequencing libraries were prepared. Paired-end sequence runs were performed with 36, 37, 54 and 76 base reads on the Illumina Genome Analyzer.
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Project description:The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged approximately 1400 bp in size when using a approximately 3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.
| S-EPMC310813 | biostudies-literature
Project description:The goals of this study are to comprehensively identify genes controlled by Jmjd6 in the thymic stroma, and to identify a novel alternative splicing mechanism. Methods: Samples were WT and Jmjd6-/- fetal thymus organ culture with (2 samples for each category) or without (1 sample for each category) RANKL stimulation for 4 days under 2-DG. One µg of total RNA was used for library construction with TruSeq RNA Sample Prep Kit v2. THe ligated products were amplified using 8 cycles of PCR to generate RNA-seq library. Library integrity was verified by Bioanalyzer DNA1000 assay. Sequencing was performed in 101-bp paired-end mode using an Illumina HiSeq.Technical duplicate has done. Results: A total of 177,060,020 reads were obtained for 6 samples. Filtered reads were mapped to the UCSC mm10 using the TopHat program(v2.0.10) with the default parameters. The Cufflinks program (v2.1.1) was then used to assemble 22,448 transcripts and to calculate the fragments per kilobase of exon per million mapped fragments(FPKM) values, which are normalized measurement of gene expression levels(= genes-FPKM file).To identify differentially expressed genes, the ratio of the maximum FPKM to the minimum FPKM was compared among 6 samples. When the ratio was more than 3, the gene was regarded as being significantly altered in expression level. We added 0.1 to the FPKM value to avoid division by zero. This led us to identify 3212 genes with differential expression. Among these, the expression levels of 2536 genes were significantly associated with the RANKL treatment or Jmjd6 expression ( P value <0.05). Analysis for intron retention was performed as follows. According to the current gene annotation ("known genes" in UCSC mm10), there are 188,208 introns in total. As intron retention events should be observed in the genes with relatively high expression, we only focused on the genes with the maximum FPKM value more than 10 at least in one of the six samples. As a result, we obtained 84,708 introns. The reads mapped to these intronic regions were counted by the intersectBed program in the BEDTools utilities with -c option, and the counts are converted into the FPKM values for each intron (intronic FPKM). There are 1051 introns with intronic FPKM more than 10 in at last on of the six samples, and the degree of intron retention was calculated by dividing intronic FPKM value by conventional FPKM value for each gene (intron-FPKM file). Samples were WT and Jmjd6-/- fetal thymus organ culture with (2 samples for each category) or without (1 sample for each category) RANKL stimulation for 4 days under 2-DG. One µg of total RNA was used for library construction with TruSeq RNA Sample Prep Kit v2. THe ligated products were amplified using 8 cycles of PCR to generate RNA-seq library. Library integrity was verified by Bioanalyzer DNA1000 assay. Sequencing was performed in 101-bp paired-end mode using an Illumina HiSeq.Technical duplicate has done.
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