|
| Status |
Public on Dec 17, 2014 |
| Title |
het_RNF17_ 5RACE_small and large500bp |
| Sample type |
SRA |
| |
|
| Source name |
adult testes of 6 week old mouse
|
| Organism |
Mus musculus |
| Characteristics |
age: 6 weeks old
genotype/variation: heterozygous RNF17 knockout
strain: C57BL/6
antibodies: none
|
| Treatment protocol |
IP with MIWI and MILI antibodies were done according (Aravin et al. 2008 Mol. Cell). Manually dissected testes were homogenized in lyses buffer and diluted 5 times in IP buffer NT2 with MIWI or MILI antibodies diluted 1:500 and incubated for 10 hours at 4C. Then protein A agarose beads (Roche) were added and incubated for 2 more hours at 4C. After 5 washes with NT2 buffer total RNA from immunoprecepitates was isolated by proteinase K treatment with following Phenol/Chloroform pH 4.8 (Ambion) extraction.
|
| Growth protocol |
mice were maintained according to the guidelines of the Cold Spring Harbor Laboratory Institutional Animal Care and Use Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted from manually dissected testes with Trizol reagent (Invitrogen)
5'RACE libraries were prepared according (Karginov et al. 2010, Mol Cell 38: 781-788). Poly(A)+ mRNAs were isolated from 100 g of total RNA using the Invitrogen Dynabeads mRNA Direct kit. The RNA was ligated to a 5 linker with T4 RNA ligase (Ambion). Reverse transcription reactions were carried out by handom hexamer priming SBS8-N6 primer and SuperScript III (Invitrogen). Template RNA was degraded by addition of 1 l RNase H (Invitrogen). PCR reactions on the resulting cDNAs were carried out using KOD Hot Start DNA polymerase (Novagen) with PE-P5-SBS3 and PE-P7-SBS8 primers. Products were run on a 2% low-melt agarose gel, and the 150500 bp (sometimes 1501000bp) range was purified.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
RACE |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Reads mapped to mm9 mouse genome release using Bowtie (Langmead et al. Genome Biol 2009, ) with up to 2 mis-matches and a maximum of 100 multiple alignments (otherwise suppressed)
Unmapped reads were re-mapped using STAR (Dobin et al.29;15-21 2013 Bioinformatics) with the same criteria to extract spliced reads (Dobin et al.29;15-21 2013 Bioinformatics). Then reads from fraction above and below 500bp were combined for the futher analysis.
Genome_build: mm9
Supplementary_files_format_and_content: txt files with all mappers, their reads number and genomic annotation
|
| |
|
| Submission date |
Jan 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Vasily Vagin |
| E-mail(s) |
vagin@cshl.edu
|
| Organization name |
Cold Spring Harbor
|
| Lab |
Hannon's
|
| Street address |
1 Bungtown Rd
|
| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE53915 |
RNF17 blocks promiscuous activity of PIWI proteins in mouse testes [5'RACE] |
| GSE53919 |
RNF17 blocks promiscuous activity of PIWI proteins in mouse testes |
|
| Relations |
| BioSample |
SAMN02571046 |
| SRA |
SRX423934 |
