ABSTRACT: Transcription profiling by array of Drosophila Kc cells after RNAi knockdown of Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) or L(3)MBT
Project description:Transcriptional profiling of C. elegans first larval stage whole animals comparing lin-35(n745) mutants and lin-35(n745) treated with mes-4(RNAi) at 26 degrees. One-condition experiment, mutant (lin-35) vs. mutant plus RNAi (lin-35, mes-4RNAi). Biological replicates 4 mutant, 4 mutant plus RNAi harvested indepedently. One lin-35 replicate and one lin-35, mes-4(RNAi) replicate per array.
Project description:Profiling of changes in steady state RNA levels upon RNAi-mediated knockdown of the chromosomal kinase JIL-1 in Drosophila S2 cells.
Project description:Profiling of changes in steady state RNA levels upon RNAi-mediated knockdown of the nucleosome remodeling ATPase ISWI in Drosophila SL2 cells. Quantitation of the corresponding proteomes has been performed to describe transcriptome-proteome relations. The analysis indicates a limited correlation of the two expression steps. Keywords: RNAi-mediated knockdown
Project description:Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNAi and micro-array expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupies thousands of chromosomal sites where a substantial number are proximal to repressed genes that are normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression whereas at other non-overlapping sites, Myb was critical for repression. These data highlight that the MMB factors are utilized in a combinatorial way for targeting gene regulation. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants. Experiment Overall Design: RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) and L(3)MBT were performed in triplicate. RNAi with a nonspecific RNA derived from a pBSK+ plasmid (named SK+) was used as control. Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (QIAGEN).
Project description:Transcriptional profiling of C. elegans first larval stage whole animals comparing lin-35(n745) mutants and lin-35(n745) treated with mes-4(RNAi) at 26 degrees.
Project description:To understand how RNAi depletion of transcription factors impact the Drosophila transcriptome, we performed selective knockdown of 46 transcription factors in Drosophila S2R+ cell line. We treated cells with long double strand RNAs by bathing for 1 to 3 days. We purified polyA+ RNA and prepared strand-specific RNA-Seq libraries. We quantitatively measured transcriptomic responses using a time series analysis.
Project description:We report the cloning and sequencing of both endogenous small RNAs and virus-derived siRNAs produced by the antiviral RNAi pathway in Drosophila. We find that a diverse panel of viruses are targeted by the RNAi pathway in Drosophila to produce abundant virus-derived siRNAs, and these siRNAs map to various locations within the viral genomes. Knockdown of various RNAi and miRNA pathway components alters the levels of these viral small RNAs.
Project description:Profiling of changes in steady state RNA levels upon RNAi-mediated knockdown of the chromosomal kinase JIL-1 in Drosophila S2 cells. Drosophila S2 cells were incubated 7 days after treatment with 10 µg of dsRNA directed against GST/EGFP or JIL-1, respectively. 5 biological replicates per target have been collected.
Project description:Purpose: Combination of systemic RNAi with RNAseq to identify target genes of important pathways in early development Drosophila early patterning occurs in the syncytial blastoderm where transcription factors diffuse between cells. However, in typical insect embryos, patterning occurs in a cellularized environment where signaling pathways are likely to play a more fundamental role. We use the short germ beetle Tribolium castaneum to investigate two putative Wnt and Hh signaling centers located in the anlagen of head and growth zone. These structures are known to develop in a different way in short germ insects. We find that Hh acts upstream of Wnt in the head, which is different from the Drosophila situation. In the growth zone Wnt signaling acts upstream. For the first time outside Drosophila, we comprehensively determine the Wnt and Hh target gene sets and distinguish the anterior from the posterior gene sets by genetically depleting head or growth zone. Surprisingly, there are significantly more targets in the growth zone than in the head for both pathways and we find that their growth zone gene sets are essentially non-overlapping. Furthermore, several pair rule genes, Tc-caudal, Tc-twist and hindgut patterning genes are regulated by Wnt signaling. In addition, Wnt controls growth zone metabolism and cell division. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. Unexpectedly, we find the Wnt target Tc-senseless to be required for hindgut development. 10-11 h old whole embyro mRNA profiles of the following treatments: reference: wild type 100bp single read triplicates reference: wild type 50bp single read triplicates treatment: Tc-arrow RNAi knockdown 50 bp single read triplicates treatment: Tc-frizzled1/2 RNAi double knockdown 50 bp single read triplicates treatment: Tc-hedgehog RNAi knockdown 100bp single reads quadruplicates treatment: Tc-orthodenticle RNAi knockdown 100bp single reads triplicates treatment: Tc-torso RNAi knockdown 100bp single reads triplicates treatment: Tc-wntless RNAi knockdown 50bp single reads triplicates Total: 2 references, 6 treatments, 25 sequencing runs