|
| Status |
Public on Jun 20, 2017 |
| Title |
MVH +/-;P0;MILI IP;piRNAs |
| Sample type |
SRA |
| |
|
| Source name |
testis
|
| Organism |
Mus musculus |
| Characteristics |
genotype: MVH +/-
developmental stage: P0
ip antibody: MILI (mouse monoclonal) (Reuter et al., 2009;PMID:19465913)
rna: piRNAs
|
| Treatment protocol |
Mvh KI allele was created by single nucleotide mutation in the exon 16 that changes the encoded amino acid (E446Q) within the helicase catalytic motif (DEAD →DQAD). Mvh null allele was created by deletion of exon 16 in the gene. Tdrd9 KI allele was created by a point mutation (E257Q) in the ATPase motif (DEVH→DQVH). Tdrd9 null allele was created by deletion of exons 3 to 5. DsRed2 reporter mouse was already described (Homolka et al,2016;PMID:26669262).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Testicular RNAs present in endogenous MILI, MIWI2, MIWI and MVH 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) and around 150-300bp (longer RNAs) were separately sequenced on Illumina HiSeq platform (EMBL Heidelberg Gene core facility).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
MILI IP
RR374-RR383.csv
RR374_mult
|
| Data processing |
Reads were sorted into individual libraries based on the barcodes, the 3′ adapter sequences were removed and 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 developed by the Sachidanandam lab (Olson et al., 2008). Only reads perfectly matching the genome were kept for further analysis.
To analyze the reporter piRNA production, the 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 of at least 15 nucleotides were then aligned to the DsRed2-LoxP-piRNAsites-in-LacZ-LoxP-SV40pA reporter sequence (Homolka et al,2016;PMID:26669262) using bowtie (Langmead et al., 2009) allowing no mismatches.
Genome_build: Reads were mapped to mm9 or the artificial DsRed2 reporter sequence.
Supplementary_files_format_and_content: The files contain the comparison of abundances of reads mapped to the genome or mapping of the reads to DsRed2 reporter (Homolka et al,2016;PMID:26669262)
|
| |
|
| Submission date |
Mar 01, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ramesh Pillai |
| E-mail(s) |
ramesh.pillai@unige.ch
|
| 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) |
| GSE95580 |
Molecular role of RNA helicases MVH and TDRD9 in PIWI slicing-triggered mammalian piRNA biogenesis and function |
|
| Relations |
| BioSample |
SAMN06470061 |
| SRA |
SRX2606396 |
