High-resolution characterization of gene function using single-cell CRISPR tiling screen.
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ABSTRACT:
SUBMITTER: Yang L
PROVIDER: S-EPMC8249386 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.
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Project description:Accelerated brain development is a unique feature of the human species. Not only the size but also morphology, in particular the connections between frontal cortex and basal ganglia distinguish the human brain from great apes and other primates. Recent findings suggest that structural features which may be important for language acquisition are influenced by FOXP2, key regulator of CNTNAP2. CNTNAP2 is one of the largest genes in the chimpanzee genome, encompassing 2.5 Mb. It encodes a neurexin with essential roles in the vertebrate nervous system. The aim of our study was to compare the methylation patterns of CNTNAP2 in human and chimpanzee brains, assuming that epigenetic regulation is essential for brain development and human language abilities. To this end, we designed a NimbleGen tiling array covering the entire chimpanzee CNTNAP2 gene plus 0.1 Mb up- and downstream flanking sequence with an average resolution of 13 bp. Methylated DNA ImmunoPreciptation (MeDIP) was used to enrich cytosine-methylated DNA fragments for downstream analysis with high-resolution tiling arrays.
2014-02-20 | GSE52949 | GEO
Project description:Accelerated brain development is a unique feature of the human species. Not only the size but also morphology, in particular the connections between frontal cortex and basal ganglia distinguish the human brain from great apes and other primates. Recent findings suggest that structural features which may be important for language acquisition are influenced by FOXP2, key regulator of CNTNAP2. CNTNAP2 is one of the largest genes in the human genome, encompassing 2.3 Mb. It encodes a neurexin with essential roles in the vertebrate nervous system. The aim of our study was to compare the methylation patterns of CNTNAP2 in human and chimpanzee brains, assuming that epigenetic regulation is essential for brain development and human language abilities. To this end, we designed a NimbleGen tiling array covering the entire human CNTNAP2 gene plus 0.1 Mb up- and downstream flanking sequence with an average resolution of 13 bp. Methylated DNA ImmunoPreciptation (MeDIP) was used to enrich cytosine-methylated DNA fragments for downstream analysis with high-resolution tiling arrays.
2014-02-20 | GSE52948 | GEO
Project description:The fungal pathogen Histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. Here we provide the first genome-wide studies to experimentally validate its genome annotation. A functional interrogation of the Histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of H. capsulatum and related fungi. We employed a three-pronged approach to provide a functional annotation for the H. capsulatum G217B strain. First, we probed high-density tiling arrays with labeled cDNAs from cells grown under diverse conditions. These data defined 6,172 transcriptionally active regions (TARs), providing validation of 6,008 gene predictions. Interestingly, 22% of these predictions showed evidence of anti-sense transcription. Additionally, we detected transcription of 264 novel genes not present in the original gene predictions. To further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. These expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional 2,249 gene predictions. Finally, we compared the G217B gene predictions to other available fungal genomes, and observed that an additional 254 gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences. These analyses yielded a high confidence set of validated gene predictions for H. capsulatum. The transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. The data is available for interactive exploration at http://histo.ucsf.edu.
2011-08-12 | GSE31155 | GEO
Project description:BACKGROUND: The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution. RESULTS: Here, we describe a high-resolution transcription map of the S. pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results indicate that approximately 66% of the genome is expressed under our experimental conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the intergenic regions, of which 36 had no predicted function. Half of the identified sRNA sequences were found to be unique to S. pneumoniae genome. We identified eight overrepresented sequence motifs among sRNA sequences that correspond to sRNAs in different functional categories. Tiling arrays also identified approximately 202 operon structures in the genome. CONCLUSIONS: In summary, the pneumococcal operon structures and novel sRNAs identified in this study enhance our understanding of the complexity and extent of the pneumococcal 'expressed' genome. Furthermore, the results of this study open up new avenues of research for understanding the complex RNA regulatory network governing S. pneumoniae physiology and virulence.
| S-EPMC2887815 | biostudies-literature
Project description:Accelerated brain development is a unique feature of the human species. Not only the size but also morphology, in particular the connections between frontal cortex and basal ganglia distinguish the human brain from great apes and other primates. Recent findings suggest that structural features which may be important for language acquisition are influenced by FOXP2, key regulator of CNTNAP2. CNTNAP2 is one of the largest genes in the human genome, encompassing 2.3 Mb. It encodes a neurexin with essential roles in the vertebrate nervous system. The aim of our study was to compare the methylation patterns of CNTNAP2 in human and chimpanzee brains, assuming that epigenetic regulation is essential for brain development and human language abilities. To this end, we designed a NimbleGen tiling array covering the entire human CNTNAP2 gene plus 0.1 Mb up- and downstream flanking sequence with an average resolution of 13 bp. Methylated DNA ImmunoPreciptation (MeDIP) was used to enrich cytosine-methylated DNA fragments for downstream analysis with high-resolution tiling arrays. MeDIP-based CNTNAP2 methylation profiling
2014-02-20 | E-GEOD-52948 | biostudies-arrayexpress
Project description:Accelerated brain development is a unique feature of the human species. Not only the size but also morphology, in particular the connections between frontal cortex and basal ganglia distinguish the human brain from great apes and other primates. Recent findings suggest that structural features which may be important for language acquisition are influenced by FOXP2, key regulator of CNTNAP2. CNTNAP2 is one of the largest genes in the chimpanzee genome, encompassing 2.5 Mb. It encodes a neurexin with essential roles in the vertebrate nervous system. The aim of our study was to compare the methylation patterns of CNTNAP2 in human and chimpanzee brains, assuming that epigenetic regulation is essential for brain development and human language abilities. To this end, we designed a NimbleGen tiling array covering the entire chimpanzee CNTNAP2 gene plus 0.1 Mb up- and downstream flanking sequence with an average resolution of 13 bp. Methylated DNA ImmunoPreciptation (MeDIP) was used to enrich cytosine-methylated DNA fragments for downstream analysis with high-resolution tiling arrays. MeDIP-based CNTNAP2 methylation profiling
2014-02-20 | E-GEOD-52949 | biostudies-arrayexpress