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| Status |
Public on Aug 16, 2017 |
| Title |
RR244;protein: none;reporter 1355:Luc-Flam718-LacZ;OSC;PIWI IP |
| Sample type |
SRA |
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| Source name |
OSC
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: ovarian somatic cell culture
tethered protein: none
reporter: 1355:Luc-Flam718-LacZ
ip antibody: PIWI (rabbit polyclonal;PMID:26166577)
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| Treatment protocol |
For fly ovarian soma expression, a recombinant line carrying the ovarian soma-specific driver traffic jam-Gal4 (tj-Gal4) (gift of A. Pelisson), and the UASp- Reporter-5BoxB was created by classical meiotic recombination in the y[1]w[1118] genetic background. Then homozygous females yw;tj-Gal4,UASp- Reporter-5BoxB were crossed with males from each line carrying the transgene expressing one of the various fusion proteins: UASp-NHA-Armi-WT, UASp-NHA-ArmiDQAG, UASp-NHA-ArmiGNT, UASp-HA-Armi-WT, UASp-NHA-Yb or UASp-NHA-Shu. The resulting F1 female progeny of the following genotype: yw;tj-Gal4,UASp- Reporter-5BoxB/+;UASp-Fusion protein/+ was selected and analyzed for piRNA expression or protein expression in the ovaries. For fly ovarian germ line expression, each transgene allowing for the expression of fusion proteins were individually combined with the ovarian germline driver line NGT-Gal4 (gift of J. Brennecke) by classical genetic crosses using balancers of the second (CyO) and third (TM6b) chromosomes from a multiple balanced line y[1],w[1118]; If/CyO; Sb/TM6b. Females yw;NGT-Gal4/(CyO); UASp-fusion protein/(TM6b) carrying or not CyO and TM6b balancers (to avoid dominant partial sterility induced by the expression of some of the transgenes) were then crossed with UASp- Reporter-5BoxB gene and the resulting F1 progeny carrying the three transgenes (yw;NGT-Gal4/UASp- Reporter-5BoxB;UASp-fusion protein/+) was selected and analyzed for piRNA expression or protein expression in the ovaries. Alternatively, in a second set of experiments, a homozygous, fully viable and fertile recombinant line yw;NGTGal4,UASp- Reporter-5BoxB was created by meiotic recombination and crossed with each of the UASp-fusion protein lines. The resulting progeny of the following genotype yw;NGT-Gal4,UASp- Reporter-5BoxB/+; UASp-fusion protein/+ was analyzed for piRNA or protein expression in the ovaries with similar results.
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| Growth protocol |
For expression in the Drosophila ovarian somatic cell (OSC) cultures, we used the pAC5.1 vector (Life Technologies) driving expression from the fly actin promoter. For expression of either HA-tag (pAC-HA) or N-HA-tag fusions (pAC-NHA), the pAC5.1 vector was further modified to add the necessary coding sequences. The HA tag is for detection of the expressed protein and theN-peptide is for artificially tethering the fusion protein to a transcript containing BoxB sequences. OSCs were cultured in 75 cm flasks and grown to 80% confluence. Approximately 3.5x106 cells were used for each electroporation reaction using Cell Line Nucleofector Kit V (Lonza, Cat No. VCA-1003) and were plated in 6-well plate. For creating transgenic fly lines, the coding sequences for NHA- or HA-tagged fusions of Armi, Yb or Shu, and the point mutant versions were inserted into the pUASp_attB_delK10 plasmid containing the white+ gene marker. These were used for site-specific integration (BestGene, Inc) in the Drosophila genome using the PhiC31 (ΦC31) integrase-mediated transgenesis system.
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| Extracted molecule |
total RNA |
| Extraction protocol |
piRNAs present in PIWI, Aub or Ago3 complexes were isolated using indicated antibodies.
Sequencing libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Cat No:E7300) according to manufacturer instructions. The synthesized libraries were resolved on agarose gel and fragments of around 120 bp (corresponding to piRNAs) sequenced on Illumina HiSeq platform (EMBL Heidelberg Gene core facility).
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
piRNAs
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| Data processing |
Reads were sorted into individual libraries based on the barcodes and the 3′ adapter sequences were clipped using cutadapt (DOI:http://dx.doi.org/10.14806/ej.17.1.200). Only reads which are at least 15 nucleotides in length were left for subsequent analysis.
Reads were then aligned to the desired reporter sequence using bowtie allowing no mismatches.
Genome_build: Reads were mapped to the reporter sequences.
Supplementary_files_format_and_content: The files contain mapping of the reads (piRNAs) to the reporter sequence.
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| Submission date |
Jul 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ramesh Pillai |
| E-mail(s) |
ramesh.pillai@unige.ch
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| Organization name |
University of Geneva
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| Department |
Department of Molecular Biology
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| Street address |
30, Quai Ernest-Ansermet
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| City |
Gneveva |
| ZIP/Postal code |
CH-1211 |
| Country |
Switzerland |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE102013 |
Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries |
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| Relations |
| BioSample |
SAMN07422154 |
| SRA |
SRX3045527 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2721800_bwt_RR244.txt.gz |
343.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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