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Sample GSM1405300 Query DataSets for GSM1405300
Status Public on Dec 22, 2014
Title Input DNA Control Day0 Replicate 2
Sample type RNA
 
Source name C2C12 Cells_Day0
Organism Mus musculus
Characteristics cell line: C2C12
cell type: myoblast
gender: female
time point: Day 0 Growing Cells
Growth protocol C2C12 mouse myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% serum (Invitrogen) and antibiotics (Invitrogen). Differentiation was induced on sub-confluent cultures by replacing the growth media (GM, DMEM with 10% FBS) with differentiation media (DM, DMEM with 2% horse serum).
Extracted molecule total RNA
Extraction protocol At various time points after differentiation, cells were harvested and RNA was extracted with phenol-chloroform and either saved as input samples for microarrays or subjected to AUF1 or IgG ribonucleoprotein immunoprecipitation. Total RNA from the various time points was either saved as examples of input RNA, or subjected to AUF1 or IgG ribonucleoprotein immunoprecipitation. C2C12 cells cultured in GM and DM were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40 for 10 min on ice and centrifuged at 15,000 × g for 10 min at 4°C. The supernatants were incubated with protein-A Dynabeads beads coated with anti-AUF1 (Millipore) or with control IgG (Santa Cruz Biotechnology) antibodies for 2 hr at 4°C. The beads were washed with NT2 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), followed by incubation with 20 units of RNase-free DNase I for 15 min at 37°C to remove the DNA. The samples were then incubated for 15 min at 55°C with 0.1% SDS/0.5 mg/ml Proteinase K to digest proteins. The RNA from the IP samples was extracted using phenol-chloroform, precipitated, and used along with the RNA from the Input samples for cDNA microarrays.
Label biotin
Label protocol Standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
 
Hybridization protocol Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
Description Input RNA Control
Input RNA Control Day0 Replicate 2
Data processing Data was extracted using the Illumina Genome Studio software, ver 1.6.0. Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores will be included in the supplemental file.
 
Submission date Jun 05, 2014
Last update date Jun 22, 2020
Contact name Supriyo De
Organization name NIA-IRP, NIH
Department Laboratory of Genetics and Genomics
Lab Computational Biology & Genomics Core
Street address 251 Bayview Blvd
City Baltimore
State/province Maryland
ZIP/Postal code 21224
Country USA
 
Platform ID GPL6885
Series (1)
GSE58259 RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels

Data table header descriptions
ID_REF
VALUE Z-score normalized signal
Detection Pval

Data table
ID_REF VALUE Detection Pval
ILMN_1212607 -0.594831578 0.30827
ILMN_1212612 -0.483466875 0.04386
ILMN_1212619 1.705679538 0
ILMN_1212628 -0.603543106 0.33835
ILMN_1212632 -0.429270355 0.0213
ILMN_1212636 2.457185909 0
ILMN_1212637 0.685201682 0
ILMN_1212645 -0.811399454 0.98747
ILMN_1212648 0.875447408 0
ILMN_1212653 0.245166048 0
ILMN_1212672 0.604627762 0
ILMN_1212682 -0.689461987 0.73684
ILMN_1212683 -0.685647955 0.72556
ILMN_1212685 -0.581052504 0.24812
ILMN_1212692 -0.494362909 0.05639
ILMN_1212693 0.259756894 0
ILMN_1212695 -0.584479121 0.26065
ILMN_1212698 -0.628356344 0.47494
ILMN_1212702 2.294703999 0
ILMN_1212703 -0.234686923 0.00125

Total number of rows: 25697

Table truncated, full table size 793 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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