Project description:Purpose: To analysis Ova impact on transcription in Drosophila ovaries by mRNA-seq and small RNA-seq Methods: mRNA-seq and small RNA-seq of RNA extracted from Drosophila ovaries Results: Ova has no obvious impact on protein coding genes expression or piRNA biogenesis.
2019-01-14 | GSE104924 | GEO
Project description:Small RNA seq of Drosophila ovaries expressing FHVdlB2eGFP replicon
Project description:Purpose: To identify significantly different individual mRNA species or genetic pathways in the GCNAKO mutant sample when compared to the wildtype sample. GCNA is encoded by CG14814 (Flybase ID: FBgn0023515). Method: Total RNA was extracted using the Qiagen miRNEasy kit, followed by on column DNAse digest using the Qiagen DNAse digest kit. We used the miRNEasy kit to ensure collection of small RNA species as well as total RNA. RNA was collected from three independent bological replicates of freshly dissected Drosophila ovaries ( 100 ovaries per replicate), collected from 1-3 day old female Drosophila. Ovaries were flash frozen in liquid nitrogen after dissection to preserve the RNA species and prevent degradation. RNAse free conditions were maintained throughout the extraction protocol. RNA quality was tested by the sequencing core before sequencing the RNA.
2019-10-22 | GSE127220 | GEO
Project description:mRNA-seq and miRNA-seq of Drosophila CG1239 knockdown ovaries
Project description:The piRNA pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and polyA tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters bypass canonical mRNA features to be specifically exported via Nxf3, ensuring proper piRNA production
Project description:Belle has been known to be co-localized with piRNA-related proteins at the nuage of germline cells during Drosophila oogenesis. However, its role in piRNA biogenesis remains unclear. To reveal whether Belle is involved in regulating piRNA expression, we performed next-generation sequencing analysis of small non-coding RNAs on ovaries harvested from the wild type (W1118) and trans-heterozygous bel[74407/neo30] mutant. Small RNA-seq experiments were performed on three individual ovary samples with the same genotype. For piRNA expression analysis, we performed mapping of three sets of small RNA sequencing reads for each genotype to previously identified eight distinct piRNA clusters located in four different Drosophila chromosomes (from X to 4). Analysis of the piRNA expression profiling from these piRNA cluster loci indicates that some specific piRNA populations were either upregulated or downregulated in bel mutant ovaries compared with wild-type ovaries. Furthermore, we performed systematic analysis by mapping piRNA sequencing reads to sequences of all identified Drosophila transposable elements (TEs) to classify and measure piRNA reads based on their TE targets. Among 124 TE-classified piRNA populations, 9 and 20 of them were upregulated and downregulated (≥2 folds), respectively, in bel74407/neo30 mutant ovaries compared with those from wild-type ovaries. To examine the effect of the bel[74407/neo30] mutation on the ping-pong cycle for secondary piRNA biogenesis, analysis of the ping-pong signature of piRNAs specifically mapped to the retro-element Burdock was performed. The ping-pong signature for the generation of secondary piRNAs was not significantly altered in bel mutants compared with the wild type. These results, taken together, indicate that Bel is not required for primary and secondary piRNA biogenesis, but it is involved in regulating expression of specific subsets of piRNA populations.
Project description:We analyzed gamaH2Av ChIP-seq from hand dissected stage 10B and 13 follicle cell nuclei. Egg chambers were dissected from wild-type (OrR) or H2Av[ΔCT] ovaries to assess binding at the Drosophila Follicle Cell Amplicons and across the genome. ChIP-seq of gammaH2Av bound to follicle cell DNA from stage 10B and 13 egg chambers, collected from wild-type (OrR) and H2Av[ΔCT] Drosophila ovaries. Sequences analyzed by Illumina sequencing. Two replicates are included for each ChIP reaction.