Transcriptome profiling of glioblastoma tissues and peritumoral regions by Serial Analysis of Gene Expression
ABSTRACT: In this study, we report a broad analysis of central tumor samples (C), from both Glioblastoma long term survivors (LT) and short term ones (ST), integrated by the same analysis performed on peritumoral areas (P) from the same patients. We provide data from SAGE analysis performed with deep sequencing.
Project description:Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor. Its prognosis is inexorably unfavorable, as these tumors drive affected patients to death usually within 15 months after diagnosis (short term survivors, ST), with the only exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Even after the frontline therapeutic approach, including surgical resection followed by chemo- and radiotherapy, the cause of death in most cases is tumor recurrence, which occurs in peritumoral tissues in about 95% of patients. Here, we provide a comprehensive molecular analysis of a set of ST and LT samples derived from frankly tumoral areas (C) and from the peritumoral regions (P) of the same patients. By performing microRNA deep sequencing, we collected data showing that P areas differ from healthy white matter, but share with C samples, a number of microRNAs
Project description:Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. Altered miRNA levels have been reported in human cancers, including RMS. Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Bioinformatics pipelines were used for miRNA target prediction and clustering analysis. Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS.
Project description:Chromatin immunoprecipitation from tammar wallaby pouch young cells for DNA bound to CENP-A Chromatin from one Tammar wallaby cell line, total of two technical replicates, 1/8th plate each, IgG and No Antibody IPs performed but not sequenced
Project description:Small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) are high-grade pulmonary neuroendocrine tumors. The neural basic helix-loop-helix (bHLH) transcription factors ASCL1 and NEUROD1 have been shown to play crucial roles in promoting the malignant behavior and survival of human SCLC cell lines. In this study, we find ASCL1 and NEUROD1 identify distinct neuroendocrine tumors, bind distinct genomic loci, and regulate mostly distinct genes. ASCL1 and NEUROD1 are often bound in super-enhancers that are associated with highly expressed genes in their respective SCLC cell lines suggesting different cell lineage of origin for these tumors. ASCL1 targets oncogenic genes such as MYCL1, RET, and NFIB, while NEUROD1 targets the oncogenic gene MYC. Although ASCL1 and NEUROD1 regulate different genes, many of these gene targets commonly contribute to neuroendocrine and cell migration function. ASCL1 in particular also regulates genes in the NOTCH pathway and genes important in cell-cycle dynamics. Finally, we demonstrate ASCL1 but not NEUROD1 is required for SCLC and LCNEC tumor formation in current in vivo genetic mouse models of pulmonary neuroendocrine tumors RNA-seq analysis performed on two ASCL1high and two NEUROD1high human SCLC cell lines to identify gene expression patterns in these cells. Also, we performed RNA-seq in mouse neuroendocrine lung tumors obtained from Trp53;Rb1;Rbl2 triple knockout model mice treated with Adeno-CMVCRE intratracheally.
Project description:Maternal diabetes is a teratogen that can lead to neural tube closure defects in the offspring. We therefore sought to compare gene expression profiles at the site of neural tube closure between stage-matched embryos from normal dams, and embryos from diabetic dams. Neurulation-stage mouse embryos at 8.5 days of gestation were used to prepare neural tissue at the anterior aspect of neural tube closure site 1. Tissue was procured from the open neural tube immediately anterior of the closure site, and from the closed neural tube immediately posterior to the closure site by laser microdissection. For each sample, 10 sections were pooled, total RNA was extracted, and 7 ng of total RNA were used for expression profiling by Tag sequencing using an Applied Biosystems SolidSAGE kit for library construction, and an AB SOLiD 5500 XL instrument for sequencing. Sequence reads were mapped to RefSeq RNA, and count data per gene were obtained using a modified version of the Applied Biosystems SOLiD™ SAGE™ Analysis Software. diabetic dam - closed neural tube // diabetic dam - open neural tube // normal dam - closed neural tube // normal dam - open neural tube
Project description:The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms of chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution, the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extra-telomeric sites contains interstitial telomeric sequences (or ITSs). However we also identified non-ITS sites, which are also satellite DNA but the ones mainly constitutive of centromeric and pericentromeric regions. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that, by binding to extratelomeric sequences, TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks. ChIP-SEQ experiment of transformed human fibroblast BJ cells with 3 antibodies (1 monoclonal anti-TRF1, 1 monoclonal anti-TRF2, 1 polyclonal anti-TRF2) and a negative control (proteinG without antibody used as the ChIP background)
Project description:mRNA seq based approach to determine the transcriptome dynamics during early development To study the mechanisms regulating this developmental event in zebrafish, we applied RNA deep sequencing technology and generated comprehensive transcriptome profiles of 6 developmental stages from oocyte to early gastrulation. We determined the expression levels of maternal and zygotic transcripts and clustered them based on expression pattern. We identified a large number of novel transcribed regions in un-annotated regions of the genome, as well as splice variants with an estimated frequency of 40-75% during early zebrafish embryogenesis. Our data constitute a useful resource for developmental studies, gene discovery, and genome annotation. RNA was extracted from pooled embryos of desired stages and one RNA seq library was generated for each sample. Totally 6 stages were selected: Maternal, 1cell, 16/32 cells, 128/256 cells, 3.5hpf and 5.3hpf
Project description:Strand specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3’ termination and bi-directional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent “horsetail” stage of meiosis. Antisense production had no impact at any of these loci when the RNAi machinery was removed. Thus, far from being simply ‘genome chatter’, this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe. Thorough interrogation of the Schizosaccharomyces pombe transcriptome during sexual differentiation using strand-specific total RNA sequencing (AB SOLiD 3.0 and 3.0+). A total of 19 samples were analysed by two separate machine runs (henceforth first and second runs, respectively). In the first machine run the following 5 samples were processed (on a single sequencing slide): Vegetative haploid (strain IH5974), pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following temperature shift from 25ºC to 32ºC to induce meiosis by Pat1 inactivation. In the second machine run the following 14 samples were processed (on two sequencing slides): Vegetative haploid (IH5974), pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following the temperature shift (a biological replicate of the first machine run). In addition, asynchronous IH3365 (wild type diploid) was also sequenced to enable a series of pair-wise haploid/diploid comparisons between itself, asynchronous haploid (IH5974) and pat1.114 diploid (IH2912) at vegetative growth. Finally, to find putative targets of the two bzip transcription factors atf21 and atf31, we sequenced RNA extracts from IH8832 (atf21.delta diploid) and IH8814 (atf31.delta diploid) before (0), and 3, 5, and 10 hours after the temperature shift, while the pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following the temperature shift were used as reference for this analysis.