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Sample GSM2896325 Query DataSets for GSM2896325
Status Public on Dec 19, 2018
Title Mouse 1- 2D- female
Sample type RNA
 
Source name 2D tissue culture of tibia and femur bone marrow stromal cells after 4 weeks of adipogenic differentiation
Organism Mus musculus
Characteristics strain: C57BL/6J
gender: female
sample type: 2D bone marrow cell
Treatment protocol See growth protocol.
Growth protocol Cells were cultured in basal growth media (DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic) for 12 days and then switched to an adipogenic differentiation media (basal supplemented with 0.5 mM IBMX, 1 uM Rosi, 1 uM Dex, 10 ug/mL insulin) for 4 days, and then a seocnd adipogenic media (basal supplemented with 1 uM Rosi and 10 ug/mL insulin) for 3 days and then maintained in a third adipogenic media (basal supplemented with 10 ug/mL insulin only).
Extracted molecule total RNA
Extraction protocol After 4 weeks of adipogenesis, total RNA was isolated from 10 samples using the Qiazol and miRNeasy isolation mini-kit (Qiagen) according to the manufacturer’s protocol. 100ng of RNA was used to synthesize cDNA through a first-strand reverse transcription reaction and second-strand RNA degradation reaction, using reagents from the GeneChip® WT PLUS Reagent Kit. cRNA was then synthesized through an overnight (16-hour) In-vitro transcription reaction, which utilizes a T7 RNA polymerase. The cRNA was purified using an Affymetrix® magnetic bead protocol. Sample concentrations were determined using a 40ug/mL/A260 constant on a Nanodrop 1000 spectrophotometer.
Label Biotin
Label protocol Approximately 5.5ug of single-stranded cDNA was fragmented using UDG (10U/uL) and APE1 (1000U/uL), provided in the GeneChip® WT PLUS Reagent Kit. Samples were then labeled with biotin using TdT (30U/uL), also provided in the GeneChip® WT PLUS Reagent Kit. Efficiency of the fragmentation and labeling reactions were verified using NeutrAvidin (10mg/mL) with a gel-shift assay.
 
Hybridization protocol Samples were combined with a hybridization mix, injected into Mouse ClariomTM S arrays, and place in the Affymetrix® GeneChip® Hybridization Oven 645 at 45° C and 60 RPM for 16.5 hours overnight.
Scan protocol Arrays were stained using the Affymetrix® GeneChip® Fluidics Station 450 and scanned with the 7G Affymetrix® GeneChip® Scanner 3000. Raw image analysis was performed with Affymetrix® Expression ConsoleTM software. The raw data images produced from the scanner were processed into .CEL files, which contain measure intensities for each probe on the array.
Description This is the first of five biological replicates of 2D bone marrow cell cultures from 16 week-old, C57BL/6J mice tibia and femurs.
Data processing The .CEL files were imported into Partek® Genomics SuiteTM (GS) version 6.6 (Partek Inc., St. Louis, MO, USA, www.partek.com). Background correction, quantile normalization, log2 transformation, and probe set summarization were performed using default settings for the Robust Multichip Average (RMA) procedure (see Bolstad et al. Bioinformatics 19(2):185; Irizarry et al. Biostatistics 4(2):249; Nucleic Acids Res. 31:e15). A two-way Analysis of Variance (ANOVA) with factors matrix type and subject (paired design) was performed on the RMA-normalized probesets to assess differential expression.
 
Submission date Dec 20, 2017
Last update date Dec 19, 2018
Contact name Heather Elizabeth Driscoll
Organization name Norwich University
Department Biology
Street address 158 Harmon Drive
City Northfield
State/province VT
ZIP/Postal code 05663
Country USA
 
Platform ID GPL23038
Series (1)
GSE108374 Development of a 3D Bone Marrow Adipose Tissue Model

Data table header descriptions
ID_REF
VALUE RMA-normalized signal intensity

Data table
ID_REF VALUE
TC0100000014.mm.2 9.48654
TC0100000018.mm.2 4.72763
TC0100000021.mm.2 7.91839
TC0100000022.mm.2 5.5993
TC0100000023.mm.2 8.75161
TC0100000027.mm.2 9.76355
TC0100000038.mm.2 8.08447
TC0100000039.mm.2 6.31098
TC0100000041.mm.2 4.8596
TC0100000044.mm.2 9.08807
TC0100000045.mm.2 3.40453
TC0100000048.mm.2 6.5694
TC0100000055.mm.2 4.30184
TC0100000056.mm.2 4.62184
TC0100000064.mm.2 9.37299
TC0100000071.mm.2 2.59559
TC0100000082.mm.2 4.66615
TC0100000092.mm.2 7.81343
TC0100000097.mm.2 6.43058
TC0100000101.mm.2 7.32259

Total number of rows: 22206

Table truncated, full table size 562 Kbytes.




Supplementary file Size Download File type/resource
GSM2896325_m12D_Reagan_9-15-2016_WTPlus_100nginput_Clariom_S_Mouse_.CEL.gz 1.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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