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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 09, 2015 |
| Title |
OSCs transfected with construct: flam promoter - flam-1624 to +112 - luciferase, Piwi IP [RR192] |
| Sample type |
SRA |
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| Source name |
OSCs
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: transfected OSCs
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| Treatment protocol |
OSCs and BmN4 cells were transfected with specific reporters. Transgenic flies were created by knocking-in the piRNA reporter sequence into Chr3 via ΦC31-mediated integration (Genetic Services Inc., USA). The pi-Chr17-ΔMyb mice was generated using Cas9-sgRNA mediated genome editing system, in collaboration with Taconic Artemis company, resulting in a deletion of chr17: 27461948 -27462175 region (mm9).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For preparation of small RNA libraries total RNA was isolated from the lysates or RNA was immunoprecipitated from the protein complexes with the indicated antibodies against PIWI protein and the RNAs were resolved by 15% or 20% urea-PAGE. Bands corresponding to piRNAs or the indicated size were excised from the gel and extracted with 400 µl of 0.3M NaCl solution at 25°C overnight. For sequencing mouse long RNAs, testicular RNAs were isolated by TRIZOL.
Small RNA sequencing libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Cat No:E7300) according to manufacturer instructions. For mouse total RNA libraries strand-specific RNA-seq libraries were prepared using TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero (mouse) (Ilumina). Libraries were sequenced on Illumina HiSeq platform (EMBL Heidelberg Gene core facility)
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
small RNA
Piwi IP
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| Data processing |
Reads were sorted into individual libraries based on the barcodes and the 3’ adapter sequences were clipped from the reads using cutadapt (DOI:http://dx.doi.org/10.14806/ej.17.1.200). Reads were then aligned to the specific reporter sequence using bowtie (Langmead et al., 2009) allowing no mismatches.
For the mouse libraries the reads were sorted into individual libraries based on the barcodes and 3’ adapter was trimmed. Reads were mapped to the mouse genome mm9. The software used for processing the data (genomic coordinates etc) from the raw data files are in-house tools from Sachidanandam lab. They are described in a publication: Olson, A.J., Brennecke, J., Aravin, A.A., Hannon, G.J. & Sachidanandam, R. Analysis of large-scale sequencing of small RNAs. Pac Symp Biocomput, 126-36 (2008).
genome build: mm9 for mouse libraries, the reads from insect libraries were mapped to specific transfected or knocked-in reporter sequence
processed data files format and content: Mouse proccessed files contain mapping of the reads to mouse genome (mm9). The processed files for insect libraries contain mapping of the reads to specific reporter sequence which was transfected into the cells or knocked-in into the fly genome .
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| Submission date |
May 21, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ramesh Pillai |
| E-mail(s) |
[email protected]
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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) |
| GSE69102 |
RNA elements in precursors instruct directional and processive primary piRNA biogenesis |
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| Relations |
| BioSample |
SAMN03703543 |
| SRA |
SRX1034997 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1692899_bwt_RR192.txt.gz |
42.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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