Project description:Canton S flies (as wild type) were bred at 25C and tightly staged 2-hr embryos were collected after 2 times of 2-hr prelay. The embryos were aged on the apple juice plates and aged to 4-6 and 6-8 hr. For each time point, two independent Chromatin immunoprecipitation (ChIP) experiments were performed using two different antibodies and compared to two independent mock ChIPs using the corresponding pre-immune serum. One Zfh1 antibody was generated in Guinea pig against full-length Zfh1 in the Furlong laboratory, and the other was a generous gift from Dr. Ruth Lehmann. ChIPs were optimized using the enrichment of a binding site identified in a pilot experiment of Zfh1- ChIP-on-chip measured by real-time PCR as previously described. The quality of each IP was also assessed by real-time PCR. Solexa libraries were prepared according to the manufacturer's recommendations. Library quality was assessed on a 2100 Bioanalyzer system (Agilent). All samples were single-end sequenced with 36-bp reads using an Illumina Genome Analyzer IIX by the EMBL Genomics Core Facility.
Project description:Whole genome sequencing of 8 F1 Drosophila lines along with the two parental lines for one of the F1 genotypes. Data were sequenced to verify previously published genome sequences (parental lines: DGRP, maternal line: PMID31308546) and to identify potentially unbalanced SNPs within the data that might confound allele-specific measurements in the F1 lines.
Project description:In this study we use a combination of proteomics Label-Free quantification methods to monitor protein expression changes over a time course of more than 20 hours of embryo development in Drosophila melanogaster.
Project description:Expression profiling of Drosophila melanogaster halo[AJ] snail[Df(2L)TE116GW11] and halo[AJ] sna[V2] homozygous mutant embryos at two timepoints of embryogenesis. Embryos were manually sorted and assayed at cellularisation stage (stage 5 to early stage 6) and at gastrulation stage (late stage 6 to early stage 8). Homozygous mutant embryos were sorted based on their reduced transparency caused by the recessive mutation of halo[AJ]. Stage-matched halo[AJ] embryos served as wildtype control. Total RNA was extracted, amplified and hybridized to Affymetrix GeneChip Drosophila Genome array version 2. Three biological replicates at each condition were generated.
Project description:Genomewide mapping of Drosophila Lmd protein binding during embryonic development. Two consecutive timepoints (6-8 and 8-10 hrs after egg-laying) were assayed in four independent repeats each. Two different antibodies were used to precipitate the Lmd protein. Additionally the respective preimmune-serum was used as a control for every precipitation. The enriched DNA was hybridized to tiling arrays covering ~ 50% of the Drosophila melanogaster genome.
Project description:CUTnTag against Beaf-32, CP190 and CTCF was performed in wild-type and insulator-depleted embryos collected between 2h10 and 2h40 after egg laying (corresponding to the stage NC14). Spike-ins from D. virilis were used in each reaction. For each sample, 50k nuclei from each species were mixed together at the start of the protocol. For each protein, CUTnTag was done in biological duplicates. Sequencing was performed on Illumina NextSeq 500.
Project description:ChIP against Opbp followed by next generation sequencing of Drosophila melanogaster embryos. Samples were sequenced using Illumina HiSeq and include four biological replicates, with both mock and input controls.
Project description:Here we improved BiTS-ChIP (Bonn et al, Nature Protocols 7, 978-994 (2012)) to identify active enhancer and promoter elements genome wide in the 104 cardiomyocytes that constitute the Drosophila heart tube and represents only ~0.5% of the total cell content of the embryo. A transgenic Drosophila strain expressing nuclear GFP under the control of a cardiac specific enhancer (TinC*>GFP) was used for staged embryo collections at stages 13-14 (10-13h of development). After embryo fixation and dissociation, intact fixed nuclei were fluorescent labelling.  Purification of this rare nuclear population was achieved by a two-step sorting procedure, yielding ~98% purity. Chromatin was extracted and used for immunoprecipitation and sequencing (ChIP-seq) to analyze chromatin modifications at promoters (H3K4me3 and H3K27ac) and enhancers (H3K27ac).  Two independent biological replicates (from FACS sorting, chromatin preparations and ChIP-Seq) were performed for each mark and sequenced using Illumina HiSeq.
Project description:Identification of translating small open reading frames (sORFs) through deep sequecing of ribosome-associated poly-adenylated RNA and conservation analysis in early Drosophila embryo
Project description:Background: The early stages of D. melanogaster embryogenesis involve cell migration and pattern formation, and lead to the formation of three germ layers (the ectoderm, mesoderm and endoderm). These developmental events are controlled by differential gene activity. In the current study we used a suppressive subtractive hybridization (SSH) procedure to identify a group of genes potentially involved in D. melanogaster early embryogenesis and to study the temporal activity of developmentally regulated genes at five different intervals covering 12 stages of embryogenesis. Results: Macroarrays were constructed to confirm induction of expression and determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes that significantly increased their expression levels at least at one developmental interval compared with the reference interval. 53.4% of them have a phenotype and/or molecular function reported in the literature, whereas 46.6% are essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 30 of the transcripts and of these, 19 (63%) proved to have restricted expression patterns. 11 of the uncharacterized genes that exhibited temporal and spatially restricted patterns of expression in developing embryos encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in S2R+ cells and analyze the subcellular distribution of recombinant proteins. Conclusions: Our data provides a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis, including novel information on the temporal and spatial expression patterns of several previously uncharacterized genes. In particular, we recovered a significant number of novel genes encoding putative secreted and transmembrane proteins, suggesting new components of signalling pathways that might be incorporated within the existing regulatory networks controlling Drosophila embryogenesis; they are also good candidates for more functional targeted analyses. Keywords: Drosophila melanogaster, gastrulation, macroarray, developmental time course We generated a subtracted stage specific cDNA library representing genes differentially expressed between Drosophila melanogaster synciyial blastoderm and gastrula stages. Clones were spotted onto nylon membranes and hybridized against cDNA probes representing mRNA from embryos at five different developmental intervals, in order to obtain time course expression data covering the first 12 stages of Drosophila development. We designed macroarrays containing either 302 non-redundant cDNAs, including protein-coding sequences, ncRNA, transposons, introns and intergenic regions (membranes A, B and C) and the complete subtracted library (579 cDNAs, membrane D). Each clone was spotted in duplicate. The following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T7 and SP6 universal primers; (2) several spots of DMSO 50% (solvent used); (3) PCR amplified fragment of genes serendipity α, twist, tinman, fog and snail as positive controls, and (4) actin, tubulin, and RP49 as housekeeping, and (5) four dilutions of a PCR amplified fragment from a B. subtilis dap cDNA (ATCC; number 87486), which hybridizes to an in vitro synthesized poly(A)-RNA that was added to the embryo RNA samples (dilution 1/200) prior to the labelling process and used as spike mRNA (Kane et al., 2000).