Project description:DNA cytosine methylation is a key epigenetic mark that is required for normal mammalian development. Iterative oxidation of 5-methylcytosine (5mC) by the TET family of DNA dioxygenases generates three oxidized nucleotides: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Recent advances in genomic mapping techniques have suggested that these oxidized cytosines not only function in the process of active reversal of 5mC but also may possess unique regulatory functions in the mammalian genome.
Project description:Using RNA-seq (RNA sequencing) of ribosome-depleted RNA, we have identified 1,273 lncRNAs (long non-coding RNAs) in P493-6 human B-cells. Of these, 534 are either up- or downregulated in response to MYC overexpression. An increase in MYC occupancy near their TSS (transcription start sites) was observed for MYC-responsive lncRNAs suggesting these are direct MYC targets. MYC binds to the same TSS across several cell lines, but the number of TSS bound depends on cellular MYC levels and increases with higher MYC concentrations. Despite this concordance in promoter binding, a majority of expressed lncRNAs are specific for one cell line, suggesting a determinant role of additional, possibly differentiation-specific factors in the activity of MYC-bound lncRNA promoters. A significant fraction of the lncRNA transcripts lack polyadenylation. The RNA-seq data were confirmed on eight selected lncRNAs by NRO (nuclear run-on) and RT-qPCR (quantitative reverse transcription PCR).
Project description:CANTATAdb 3.0 is an updated database of plant long non-coding RNAs (lncRNAs), containing 571,688 lncRNAs identified across 108 species, including 100 Magnoliopsida (flowering plants), a significant expansion from the previous version. A notable feature is the inclusion of 112,980 lncRNAs that are expressed specifically in certain plant organs or embryos, indicating their potential role in development and organ-specific processes. In addition, CANTATAdb 3.0 includes 74,886 pairs of evolutionarily conserved lncRNAs found across 47 species and inferred from genome-genome alignments as well as conserved lncRNAs obtained using a similarity search approach in 5,479 species pairs, which would further aid in the selection of lncRNAs for functional studies. Interestingly, we find that conserved lncRNAs with tissue-specific expression patterns tend to occupy the same plant organ across different species, pointing toward conserved biological roles. The database now offers extended search capabilities and downloadable data in popular formats, further facilitating research on plant lncRNAs.
Project description:Various test protocols to improve the 3' pull down transcript profiling protocol, aiming to produce a pipeline library prep protocol. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ Protocol: Total RNA was extracted from zebrafish embryos using Trizol and DNase treated. Chemically fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyT oligo with part of Illumina adapter 1 at the 5 end followed by 12 random bases, then an 8 base indexing tags, then CG and 14 T bases. An Illumina library with full adapter sequence was produced by 20 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Set up and modification of Agilent standard protocols for a new custom 60-mer array for C. acetobutylicum. Keywords: Test of platform and protocols
Project description:BACKGROUND:Prostate cancer (PCa) is a complex disorder resulting from the combined effects of multiple environmental and genetic factors. Small non-coding RNAs (sRNAs), particularly microRNAs (miRNAs), regulate several cellular processes and have an important role in many human malignancies including PCa. We assessed the sRNA profiles associated with PCa in Arabs, a population that has rarely been studied. METHODS:We used next generation sequencing technology to obtain the entire sRNA transcriptome of primary prostate tumor formalin-fixed paraffin-embedded tissues, and their paired non-tumor tissues, collected from Bedouin patients (Qatari and Saudi). The miRNA and the target gene expression were evaluated by real-time quantitative PCR. miRNA KEGG pathway and miRNA target genes were subsequently analyzed by starBase and TargetScan software. RESULTS:Different expression patterns of several sRNA and miRNA editing were revealed between PCa tumor and their paired non-tumor tissues. Our study identified four miRNAs that are strongly associated with prostate cancer, which have not been reported previously. Differentially expressed miRNAs significantly affect various biological pathways, such as cell cycle, endocytosis, adherence junction and pathways involved in cancer. Prediction of potential targets for the identified miRNAs indicates the overexpression of KRAS, BCL2 and down-regulation of PTEN in PCa tumor tissues. CONCLUSION:These miRNAs, newly associated with prostate cancer, may represent not only markers for the increased risk of PCa in Arabs, but may also reflect the clinical and pathological diversity as well as the ethno-specific heterogeneity of prostate cancer.
Project description:Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these genes represented leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 10 intergenic small RNAs were mapped. 6 intergenic small RNAs, including 4.5S RNA and rnpB, were transcribed at extremely high levels. Although several intergenic sRNAs are conserved in M. avium and M. tuberculosis, both of these species have unique intergenic sRNAs. Moreover, we demonstrated that even conserved small RNAs are regulated differently in these species. Different sets of intergenic sRNAs may underlie differences in physiology between conditionally pathogenic M. avium and highly specialized pathogen M. tuberculosis.
Project description:RNA samples from human tissues (brain and liver) and cell lines (K562 and HL60) were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Technical replicates and different cDNA synthesis protocols were compared. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation.
Project description:BACKGROUND:Arrhythmogenic cardiomyopathy (ACM) is a genetic autosomal disease characterized by abnormal cell-cell adhesion, cardiomyocyte death, progressive fibro-adipose replacement of the myocardium, arrhythmias and sudden death. Several different cell types contribute to the pathogenesis of ACM, including, as recently described, cardiac stromal cells (CStCs). In the present study, we aim to identify ACM-specific expression profiles of human CStCs derived from endomyocardial biopsies of ACM patients and healthy individuals employing TaqMan Low Density Arrays for miRNA expression profiling, and high throughput sequencing for gene expression quantification. RESULTS:We identified 3 miRNAs and 272 genes as significantly differentially expressed at a 5% false discovery rate. Both the differentially expressed genes as well as the target genes of the ACM-specific miRNAs were found to be enriched in cell adhesion-related biological processes. Functional similarity and protein interaction-based network analyses performed on the identified deregulated genes, miRNA targets and known ACM-causative genes revealed clusters of highly related genes involved in cell adhesion, extracellular matrix organization, lipid transport and ephrin receptor signaling. CONCLUSIONS:We determined for the first time the coding and non-coding transcriptome characteristic of ACM cardiac stromal cells, finding evidence for a potential contribution of miRNAs, specifically miR-29b-3p, to ACM pathogenesis or phenotype maintenance.
Project description:Functional Magnetic Resonance Imaging (fMRI) has furthered brain mapping on perceptual, motor, as well as higher-level cognitive functions. However, to date, no data collection has systematically addressed the functional mapping of cognitive mechanisms at a fine spatial scale. The Individual Brain Charting (IBC) project stands for a high-resolution multi-task fMRI dataset that intends to provide the objective basis toward a comprehensive functional atlas of the human brain. The data refer to a cohort of 12 participants performing many different tasks. The large amount of task-fMRI data on the same subjects yields a precise mapping of the underlying functions, free from both inter-subject and inter-site variability. The present article gives a detailed description of the first release of the IBC dataset. It comprises a dozen of tasks, addressing both low- and high- level cognitive functions. This openly available dataset is thus intended to become a reference for cognitive brain mapping.