|
| Status |
Public on Dec 17, 2014 |
| Title |
hom RNF17 6 week old adult testis rep2 RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
adult testes of 6 week old mouse
|
| Organism |
Mus musculus |
| Characteristics |
age: 6 weeks old
genotype/variation: homozygous RNF17
strain: C57BL/6
antibody: none
|
| Treatment protocol |
IP with RNF17 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 RNF17 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 prepared with Trizol reagent (Invitrogen)
Transcriptome libraries were prepared according (Armour et al. 2009, Nat Methods) using not-so-random priming (NSR). 1 ug total RNA was revere transcribed using SuperScript III enzyme with first-strand NSR primer. RNA template was removed with RNase H (Invitrogen) Then cDNA was mixed with exo− Klenow fragment (NEB) second-strand NSR primer to synthesize the second strand. For PCR amplification, purified second-strand synthesis reaction were mixed with ExpandPLUS enzyme (Roche) and PE-P5-SBS3 and PE-P7-SBS8 primers. Products were run on a 2% low-melt agarose gel, and the 350–500 bp. range were purified.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
First 8 nucleotides from 5 'end were trimmed and reverse-complemented before genomic mapping
Mapped to mm9 mouse genome release with STAR (Dobin et al.29;15-21 2013 Bioinformatics) with 2 mismatches and maximum of 100 multiple alignments.
DESeq was used according to (Anders and Huber 2010, Genome Biol 11: R106.)
Genome_build: mm9
Supplementary_files_format_and_content: results of DESeq analysis only for transcripts which are deferentially expressed between homozygous and heterozygous RNF17mutant 6 week old adult testes include fold change in raw counts normalized according to DESeq protocol (Anders and Huber 2010, Genome Biol 11:R106) (up- (fold change<0) or down-regulated (fold change>0)) and p-values. Number of reads that map to genes or uniquely or multiple times (2-99) to tranposable elements form RNF17 IP or total testes lysate.
|
| |
|
| 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) |
| GSE53913 |
RNF17 referees ping-pong in mouse testes [RNAseq] |
| GSE53919 |
RNF17 blocks promiscuous activity of PIWI proteins in mouse testes |
|
| Relations |
| BioSample |
SAMN02571037 |
| SRA |
SRX423929 |
