|
| Status |
Public on Dec 04, 2015 |
| Title |
Exd1 +/- ; MILI IP rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Exd1 +/-_MILI IP
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6;129S4/SvJae mixed
genotype/variation: Exd1 +/-
developmental stage: P0
tissue: testis
ip antibody: MILI (mouse monoclonal); PMID 19465913
molecule subtype: small RNA
|
| Treatment protocol |
Exd1 null allele was made by an insertion of an in-frame triple stop codon cassette immediately downstream of the first codon in the exon 8. Rosa26-pi reporter mouse was made by insertion of reporter sequence into Rosa26 locus. The reporter sequence consists of the DsRed2 coding sequence, the 3′ UTR based on the LacZ backbone with binding sites for 35 MILI-bound piRNAs, flanking LoxP sites, SV40 polyA signal, FRT-flanked Keo reporter cassette (with its own polyA signal).
|
| 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% urea-PAGE. Bands corresponding to piRNAs or the indicated size were excised from the gel and extracted with 400 µl of 0.4M NaCl solution at 25°C overnight.
Small RNA sequencing libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Cat No:E7300) according to manufacturer instructions. Libraries were sequenced on Illumina HiSeq platform (EMBL Heidelberg Gene core facility).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
MILI IP
processed data file: Exd1_mappers.csv.gz
RR215
|
| Data processing |
Reads obtained from Rosa26-pi reporter mouse libraries 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 Exd1 libraires the barcodes were removed, and 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 [PMID 18229681]
Genome_build: mm9 for Exd1 libraries, the reads from Rosa26-pi reporter mouse libraries were mapped to the reporter sequence.
Supplementary_files_format_and_content: Exd1 proccessed files contain mapping of the reads to mouse genome (mm9). The processed files for Rosa26-pi reporter mouse libraries contain mapping of the reads to the specific reporter sequence knocked-in into the mouse genome .
|
| |
|
| Submission date |
Oct 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ramesh Pillai |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Geneva
|
| Department |
Department of Molecular Biology
|
| Street address |
30, Quai Ernest-Ansermet
|
| City |
Gneveva |
| ZIP/Postal code |
CH-1211 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE74423 |
PIWI slicing and EXD1 drive biogenesis of nuclear piRNAs from cytosolic targets of the mouse piRNA pathway |
|
| Relations |
| BioSample |
SAMN04219609 |
| SRA |
SRX1392301 |
