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Sample GSM566422 Query DataSets for GSM566422
Status Public on Jul 15, 2010
Title Endougenous small RNA from MHV-68 infected mouse S11 cell, treated with TPA
Sample type SRA
 
Source name mouse B lymphoma S11 cells, treated with TPA
Organism Mus musculus
Characteristics cell type: mouse B lymphoma
cell line: S11
Treatment protocol S11 cells were treated with TPA (20 ng/ml) for 48h.
Growth protocol S11 cells were cultured in RPMI 1640 (Cell Concepts, Umkirch, Germany) supplemented with 10% FCS, 2 mM L-Glutamine, 100 U/ml Penicillin, 100 μg/ml Streptomycin and 50 µM 2-Mercaptoethanol.
Extracted molecule total RNA
Extraction protocol From all samples, RNA species smaller than 200 bases were enriched with the mirVana miRNA isolation kit (Ambion, Austin, Texas, USA). The small RNAs from all 4 samples were separated on a denaturing 12,5% polyacrylamide (PAA) gel and stained with SYBRgreenII. As molecular mass standard a mixture of oligonucleotides was used that range in size between 15 and 30 bases. The population of miRNAs with a length of 15 – 30 bases was obtained by passive elution of the RNAs from the gel. The miRNAs were then precipitated with ethanol and dissolved in water. For cDNA synthesis the RNAs were first poly(A)-tailed using poly(A) polymerase followed by ligation of a RNA adapter to the 5´-phosphate of the miRNAs. First-strand cDNA synthesis was then performed using an oligo(dT)-linker primer and M-MLV-RNase H- reverse transcriptase. The resulting cDNAs were then PCR-amplified to about 20 ng/μl using the high fidelity polymerase Phusion (Finnzymes). The fusion primers used for PCR amplification were designed for amplicon sequencing according to the instructions of 454 Live Sciences. Barcode sequences for each cDNA species are attached to the 5'-ends of the cDNAs. cDNAs of 4 samples were mixed in equal amounts. The correct size range was obtained by separation of the mixed cDNAs on and electroelution of the 120 – 135 bp fraction from 6% PAA-gels followed by purification of the eluted cDNAs using Nucleospin Extract II (Macherey and Nagel). The cDNAs were pooled (Volume: 30 μl; Concentration: 13 ng/μl; dissolved in 5 mM Tris pH:8,5) and sent for 454 sequencing.
 
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model 454 GS 20
 
Description Total endogenous small RNAs from NPC sample of patient1
Data processing Processed data file contains the reads with 5' and 3' adapters removed. Same reads are sorted together and the numbers of repetitivity are illustrated
 
Submission date Jul 14, 2010
Last update date May 15, 2019
Contact name Gunter Meister
E-mail(s) meister@biochem.mpg.de
Organization name Max Planck Institute of Biochemistry
Department Center for Integrated Protein Science Munich (CIPSM)
Lab RNA Biology
Street address Am Klopferspitz 18
City Martinsried
ZIP/Postal code 82152
Country Germany
 
Platform ID GPL9522
Series (1)
GSE22938 Identification and analysis of expression of novel microRNAs of murine gammaherpesvirus 68
Relations
SRA SRX028227
BioSample SAMN00115711

Supplementary file Size Download File type/resource
GSM566422_S11+.txt.gz 91.7 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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