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Sample GSM566419 Query DataSets for GSM566419
Status Public on Jul 15, 2010
Title Endougenous small RNA from mouse NIH3T3 cells, without MHV-68 infection
Sample type SRA
Source name mouse fibroblast NIH3T3 cells, without MHV-68 infection
Organism Mus musculus
Characteristics cell type: mouse embryo fibroblast
cell line: NIH3T3
Treatment protocol No further treatment.
Growth protocol NIH3T3 cells (ATCC CRL-1658) were grown in DMEM High Glucose (Cell Concepts, Umkirch, Germany) supplemented with 10% FCS, 2 mM L-Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin.
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
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
SRA SRX028224
BioSample SAMN00115708

Supplementary file Size Download File type/resource
GSM566419_NIH3T3-.txt.gz 66.2 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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