Project description:This experiment contains the subset of data corresponding to human RNA-Seq data from experiment E-GEOD-30352 (http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-30352/), which goal is to understand the dynamics of mammalian transcriptome evolution. To study mammalian transcriptome evolution at high resolution, we generated RNA-Seq data (∼3.2 billion Illumina Genome Analyser IIx reads of 76 base pairs) for the polyadenylated RNA fraction of brain (cerebral cortex or whole brain without cerebellum), cerebellum, heart, kidney, liver and testis (usually from one male and one female per somatic tissue and two males for testis) from nine mammalian species: placental mammals (great apes, including humans; rhesus macaque; mouse), marsupials (gray short-tailed opossum) and monotremes (platypus). Corresponding data (∼0.3 billion reads) were generated for a bird (red jungle fowl, a non-domesticated chicken) and used as an evolutionary outgroup.
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome. 4mC-TAB-seq
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:5-hydroxymethylcytosines (5hmC) is particularly abundant in mammalian brain with little-known functions. Here we present the first genome-wide and single-base-resolution maps of 5hmC and 5mC in human brain by combined application of TAB-Seq and MethylC-Seq. We report that the majority of modified cytosines are hydroxymethylated in adult human brain, a significant proportion of which are highly-hydroxymethylated with enrichment in active genic regions and distal-regulatory elements. 5hmC is more enriched in poised than active enhancers, and CpG island shores and enhancers show comparable 5hmC profiles. Notably, 5hmC spikes were identified at the 5’ splicing sites, suggesting a link between 5hmC and splicing. Additionally, we identified a transcription-correlated 5hmC bias towards to sense strand and a 5mC bias towards antisense strand of gene bodies, and a bias towards C-rich sequences surrounding the 5hmC sites. Our data imply multiple roles for 5hmC in alternative splicing and gene regulation in addition to be an intermediate of DNA demethylation in human brain. Examination of hydroxymethylomes of 1 adult and 1 fetal brain tissue of frontal lobe, as well as 1 methylome of the same adult.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes