Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA210117 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Application of this method allows whole genome studies from samples where material or yields are limiting. Comparison of ChIP-seq libraries constructed from 100 pg DNA (this study) and nanograms of DNA (modENCODE). ChIP antibody: H3K27me3, Active Motif 31955.
2013-07-01 | E-GEOD-48431 | biostudies-arrayexpress
Project description:High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Application of this method allows whole genome studies from samples where material or yields are limiting.
2013-07-01 | GSE48431 | GEO
Project description:The 3' ends of most Drosophila melanogaster genes are poorly annotated or are determined by only a single EST or cDNA clone. To enhance the annotation of poly(A) site use in Drosophila, we performed deep sequencing on RNA isolated from 29 dissected tissues using an approach designed to enrich for poly(A) spanning reads. From these experiments, we identified 1.4 million poly(A) spanning reads leading to the identification of many new poly(A) sites and the identification of many tissue-specific poly(A) sites. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf RNA from 29 dissected Drosophila melanogaster tissues (in duplicate) were used to prepare polyA enriched RNA-Seq libraries. Briefly, total RNA was poly(A) selected, fragmented, and ligated to 5' and 3' RNA linkers. These libraries were amplified using Illumina paired-end primers, and subsequently reamplified using a 3' primer complementary to the 3' adapter but containing 6 Ts at the 3' end. The libraries were also multiplexed and up to 12 samples mixed per lane and sequenced on an Illumina GAIIx using paired-end 76 bp reads, or an illumina HiSeq 2000 using paired-end 100 bp reads. All reads were mapped to the Drosophila melanogaster genome to identify unmapped reads. Unmapped reads containing at least 10 A residues at the 3' end were identified, the terminal A residues trimmed, realigned to the genome to identify uniquely mapped reads. Such reads were identified as polyA spanning reads
2011-11-23 | E-GEOD-33905 | biostudies-arrayexpress
Project description:RNA-sequencing of twelve treatments: two cultivars (Embrapa 45 and BR 4), two oxygen conditions [fully aerobic state (normoxy) and hypoxic], and three treatment sampling times (0.5h, 4h, and 28h). For each of twelve treatments, equimolar quantities of purified total RNA from roots of twelve plants [three biological replicates (four plantlets per replicate)] were pooled to result one library. After processing the twelve libraries (poly-A purification, fractionation, cDNA synthesis using random primers, and ligation to bar-coded adapters), fragments of 150250 pb were isolated and multiplexed, resulting one sequencing library (a pooled of equimolar quantities from twelve initial libraries; each library with a specific barcode for further bioinformatic discrimination). Sequencing library was used to produce clusters for a 1 x 100 bp single end-sequencing run into one lane on a flow cell for sequencing in a Hi-Seq 2000 (Illumina).
2017-11-21 | E-MTAB-5709 | biostudies-arrayexpress
Project description:Chromatin immunoprecipitation was performed with modification of the protocols described before (Lee et al., 2006; Lilja et al., 2007). Briefly, the chorion was removed from 12-hour old embryos, cross-linked using 2% paraformaldehyde and resuspended in storage buffer (50mM Tris-HCl pH8.0, 1 mM EDTA). Embryos were then lysed in SDS-lysis buffer (1.0 ml 5 M NaCl, 2.5 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml 10 % SDS), resuspended in SDS-lysis and Triton buffer (1.0 ml 5 M NaCl, 5.0 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml Triton X-100, protease inhibitor cocktail) and sonicated on ice to an average length of 350 bp. The resulting sheared chromatin (25Izg) was subjected to immunoprecipitation using anti-Myc antibody (Santa Cruz) as described previously (Lee et al., 2006). ChIP-sequencing libraries were constructed following manufactureras instructions (Illumina). The resulting DNA libraries were sequenced using Illumina platform (University of Massachusetts Medical School Core Facility).
2019-02-02 | E-MTAB-4083 | biostudies-arrayexpress
Project description:Transcriptional profiling of anterior ovarioles (germaria and round previtellogenic egg chambers) of w1118 virgin females of Drosophila melanogaster 5 to 8 days post eclosion. Anterior ovarioles were dissected from w1118 virgin females 5 to 8 days post eclosion. Three independent biological replicates were prepared for each day. Total RNA was isolated from the samples and used for the generation of multiplexed stranded PolyA+ libraries. External RNA controls were added to ovarian polyA+ RNA during library preparation. 75bp single end libraries were sequenced by Illumina Hiseq 2000.
2016-04-04 | E-GEOD-79550 | biostudies-arrayexpress
Project description:Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: methylcytosine has been detected at very low levels in early embryos, but the genomic location and function of methylation has not been established. We have mapped cytosine methylation genomewide in Stage 5 Drosophila embryo DNA by combining immuno-enrichment for 5-methylcytosine, bisulfite conversion, and deep sequencing. Unlike methylation patterns observed in other eukaryotic species, methylation in Drosophila is punctate and highly strand-asymmetrical; we confirmed this by direct PCR amplification and sequencing of bisulfite-converted DNA. Despite the locally asymmetric nature of methylation, large-scale patterns of methylation are symmetric. Methylated regions make up ~1% of the genome, and within these regions methylation of individual cytosines averages 2-10%. Methylation is concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. It is depleted from promoters, coding sequences, and most retrotransposons, and enriched in introns and in certain simple sequence repeats containing the commonly methylated motifs. Comparison with available gene expression data indicates that methylation in a gene is associated with lower expression; the X chromosome, which is subject to gene dosage compensation, is more densely methylated than the autosomes. This study firmly establishes the presence of cytosine methylation in Drosophila; the temporal overlap of methylation with the maternal-zygotic transition raises the possibility that methylation participates in the transition to zygotic gene expression. To enrich for rare cytosine methylation in Drosophila at embryonic Stage 5 (2-3 hours post-fertilization), we enriched sonicated Stage 5 genomic DNA for methylcytosine by immunoprecipitation with antibody to 5-methylcytosine. The immunoprecipitated DNA was then bisulfite converted and Illumina sequenced to obtain direct evidence for the presence of methylation. The presence and extent of DNA methylation was confirmed by Illumina sequencing of bisulfite-converted PCR amplicons.
2014-02-18 | E-GEOD-34425 | biostudies-arrayexpress
Project description:Male flies were crossed to virgins of genotype yw;esg-Gal4,UAS-GFP;tub-Gal80ts/Tm3,Sb. Progeny was collected and aged 3-4 days before shifting for 7 days to 29 degrees to induce RNAi expression. Guts were dissected for 2 biological replicates/condition, each containing 80-100 guts, dissociated and GFP-positive cells were FACS-sorted. RNA was isolated using the PicoPure RNA-isolation kit and libraries were generated. Sequencing was done by distributing the 4 libraries over 5 lanes on the Illumina HiSeq2000 platform.
2021-04-02 | GSE163406 | GEO
Project description:To determine the genome-wide occupancy of the Plasmodium falciparum transcriptional regulator of invasion PfAP2-I (PfDd2_100013100/PF3D7_1007700), we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Synchronized, schizont stage, 40 hours post-invasion, cultures of parasites expressing the AP2-I-GFP fusion protein were treated with formaldehyde to crosslink proteins to DNA and harvested. After shearing the DNA, the chromatin was incubated with anti-GFP antibody or IgG (as control) for immunoprecipitation. This material was used to generate Illumina sequencing libraries. The final libraries were multiplexed with fourteen barcoded samples per lane on an Illumina HiSeq 2500 system to generate 150 base pair single-end reads.
2017-06-01 | GSE80293 | GEO
Project description:piRNA libraries from Drosophila melanogaster ovaries
| PRJNA98965 | ENA