Gene expression profiling of liver from C57BLKS/J and db/db 8 mice with an AAV harboring a control or GAbpa-specific miRNA under the LP1 promoter
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ABSTRACT: To initially determine changes on the transcriptome level that might account for the phenotypic observations in an unbiased fashion, we performed large-scale mRNA expression profiling 8 weeks after treatment with an AAV harboring a control or GAbpa-specific miRNA under the LP1 promoter. Mice were subjected to 16 h fasting or a 16 h fasting + 6 h refeeding cycle before termination of the experiment. All 8 AAV-injected experimental groups as NC and GAbpα knockdown samples for the different conditions (wt or db/db; fasting or refeeding) were included.
Project description:We prepared small RNA libraries from 29 tumor/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for analysis of class differences between high throughput sequencing datasets, and describe a novel application of log linear model that has provided the most datasets, and describe a novel application of log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumor and normal samples at a false discovery rate less than 0.001. A total of 29 tumor/normal pairs of human cervical tissue samples were analyzed. Two samples (G699N_2 and G761T_2) were performed in duplicates. No Fastq files for GSM532871 to GSM532889, GSM532929, and GSM532930. Sequence files are provided as text files for these 22 Sample records in GSE20592_RAW.tar. 38 samples with quality scores are available from SRA as SRP002/SRP002326 (see Supplementary file below).
Project description:The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5' end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5' uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3' terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo. We compared Twi1p-loaded scnRNAs to scnRNAs before they have been loaded into Twi1p by deep sequencing to understand how the two processes, the production of siRNAs by Dicer and the loading of siRNAs into Argonaute, shape the population of siRNAs in vivo.
Project description:The earliest recognizable stages of breast neoplasia are lesions that represent a heterogeneous collection of epithelial proliferations currently classified based on morphology. Their role in the development of breast cancer is not well understood but insight into the critical events at this early stage will improve efforts in breast cancer detection and prevention. These microscopic lesions are technically difficult to study so very little is known about their molecular alterations. To characterize the transcriptional changes of early breast neoplasia, we sequenced 3'- end enriched RNAseq libraries from formalin-fixed paraffin-embedded tissue of early neoplasia samples and matched normal breast and carcinoma samples from 25 patients. We find that gene expression patterns within early neoplasias are distinct from both normal and breast cancer patterns and identify a pattern of pro-oncogenic changes, including elevated transcription of ERBB2, FOXA1, and GATA3 at this early stage. We validate these findings on a second independent gene expression profile data set generated by whole transcriptome sequencing. Measurements of protein expression by immunohistochemistry on an independent set of early neoplasias confirms that ER pathway regulators FOXA1 and GATA3, as well as ER itself, are consistently upregulated at this early stage. The early neoplasia samples also demonstrate coordinated changes in long non-coding RNA expression and microenvironment stromal gene expression patterns. This study is the first examination of global gene expression in early breast neoplasia, and the genes identified here represent candidate participants in the earliest molecular events in the development of breast cancer. 3SEQ was performed on 72 FFPE human breast samples from 25 patients: 24 normal, 25 early neoplasia, 9 carcinoma in situ, and 14 invasive cancer
Project description:CAMPARI2 CRISPR screening for SOCE modulators of ER stress. PC cells were sorted and sequenced for CRISPR whole KO library (De brie). Unsorted, SOrte din LOW PC and LOW PC Tunica treated fro 4hours were analysed.
Project description:EBV-positive cell lines were assayed for expression of EBV miRNAs. The names of the miRNAs are from miRBase from Fall 2007. Microarray probes are tandem complements of the mature miRNA sequence. We assayed Burkitt's lymphoma (BL), Nasopharyngeal carcinoma, post-transplant lymphoproliferative disease (PTLD), primary effusion lymphoma, and lymphoblastoid cell lines. We also assayed primary B cells that were infected with the B95-8 strain of EBV, which was found to express EBV miRNAs as early as 20 hours post infection. We have found PTLD and BLs from HIV-positive donors both express EBV miRNAs. These types of cell lines have not previously been found to express viral miRNAs. We have found that cells that support type I and type II latency express only the BART miRNAs, whereas cells that support type III latency express BART and BHRF1 miRNAs. Furthermore, BL cell lines that spontaneously lose EBV express levels of the viral miRNAs that are at least 5-fold lower than cell lines that do not lose EBV. In total, 48 samples have been assayed and included in this study. EBV-negative control samples are not included in this data set, but raw and processed data may be requested from the contributors. These EBV-negative cell lines include the Burkitt's lymphoma cell lines, BJAB and Akata-negative, the gastric carcinoma cell line, AGS, and uninfected primary B cells. Of the 48 samples, we have assayed 22 different EBV-positive cell lines and 4 different time points after infection of primary B cells with EBV. Replicates of the majority of cell lines is included in this data set. Replicates are from independent RNA isolations that were then hybridized to individual microarrays.
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
Project description:Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of human cancer remains unclear. Here, we report the creation of a mouse model in which centrosome number can be persistently increased in the absence of additional genetic defects. We show that extra centrosomes increase tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive development of spontaneous tumors in multiple tissues. Tumors with centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship between centrosome amplification, genomic instability and tumor development. The sequences in this dataset result from random whole-genome DNA sequencing of spontaneous T- cell lymphomas, B-cell lymphomas, squamous cell carcinomas and one sarcoma from doxycycline-treated Plk4 mice.
Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
Project description:The 3' untranslated region (3'UTR) constitutes a major site of post-transcriptional regulation of gene expression. Sequence elements in the 3'UTR interact with trans-acting regulators such as microRNAs that affect translation and stability. The overall aim is to use a 3'RACE cloning-sequencing stragety to identify the 3'UTRs of C. elegans transcripts and explore their heterogeneity in different developmental stages and tissues. Keywords: Transcriptome analysis For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Raw data files are available on our FTP site: ftp://ftp.ncbi.nlm.nih.gov/pub/geosup/Series/GSE17781 pilot study [GSM443959..GSM443964]: N2 wildtype worms staged at embryo, L1, L2, L3, L4, and adult full experiment [GSM446651..GSM446661]: N2 wildtype worms staged at embryo, L1, L2, L3, L4, dauer, and adult. Illumina Genome Analyzer sequencing of isolated clones [GSM469439] 454 sequencing of RACE clones [GSM469976]