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Sample GSM1303493 Query DataSets for GSM1303493
Status Public on Dec 17, 2014
Title hom_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: homozygous 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
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
BioSample SAMN02571049
SRA SRX423935

Supplementary file Size Download File type/resource
GSM1303493_hom_RNF17_5RACE_processed.txt.gz 345.9 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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