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 Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts. Structural variations of three cancer cell lines using short (1 kb) and long (10 kb and 20 kb) insert size DNA fragments
Project description:Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts.
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.
