Project description:Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)Âdependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidÂbased functions and include Argonaute-2 (AGO2) itself. Experiment Overall Design: Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol. Experiment Overall Design: Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3¹ Amplification One-Cycle Target labeling kit according to manufacturer¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
Project description:In order to identify interaction partner of the Drosophila melanogaster TFIIA protein, we have immunoprecipitated an endogenously 3xFLAG-AID tagged TFIIA-L from Drosophila Schneider S2 cells
Project description:2 million cells were seeded in a 2 ml medium containing 58 µg of total dsRNA. For coRNAi treatments 28 µg of each single dsRNA were used. After treatment cells were left to incubate 4 d at 25 °C, and plates were sealed with Parafilm to avoid medium evaporation. 8 samples were prepared with the following dsRNA treatment combinations: RLUC/RLUC x 2, CSN5/RLUC, CSN8/RLUC, CDK2/CSN5, CDK2/CSN8, CDK2/RLUC 2x . dsRNAs were taken from the HD3-dsRNA-library. Two dsRNAs per gene were pooled. The single-cell transcriptomic sequencing experiment was performed with 10x Genomics technology according to the manufacturer’s protocol.
Project description:Drosophila S2 cells treated with either GFP or spottes-dick dsRNA and incubated for 5 days. There are three replicates for each condition. Keywords = Spotted-dick Keywords: other
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:The centromere-specific Histone H3-variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse-induction of CENH3 (Drosophila CID) in Schneider S2 cells incorporates into noncentromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and display a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing Histone acetylation interferes with CID islands formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1-LacI fusions to stably integrated Lac Operator arrays produces a proximal hotspot for CID islands formation. These data suggest that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.
Project description:Analysis of H3K9me3 and H3K36me3 levels in unique and repetitive regions of Drosophila genome after GFP (control) or dKDM4A RNAi of S2 cells