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Sample GSM459573 Query DataSets for GSM459573
Status Public on Nov 30, 2009
Title Wild type mouse #12
Sample type RNA
 
Source name Spleen-derived B-cells from B6 mouse
Organism Mus musculus
Characteristics strain: B6
cell type: spleen-derived B cells
genotype: Wild type
age (months): 8
developmental stage: adult
sex: Female
Treatment protocol None.
Growth protocol Six Nf1+/- B6 (three males, three females) and six wild-type (three males, three females) adult (“Nf1-Mouse-12”) mice from 5 litters of the same line were euthanized at the age of ~8 months with CO2. The dissected spleens were homogenized with the tip of a syringe plunger (3 ml syringe, slip tip, BD Biosciences, Discovery Labware, Bedford, MA, USA) and passed through a 70 μm nylon cell strainer (BD Biosciences) and purified using the CD19 mouse microbeads (Miltenyi Biotec, Auburn, CA, USA) on the autoMACS Separator (Miltenyi Biotec) using the possel_d program, following the manufacturer’s directions. CD19+ and CD19- flow-through fractions were collected, and the purity was assessed by FACS on aliquots using GR1-FITC, B220-PE, CD3-PerCPCy5.5 and MAC1-APC antibodies (BD Pharmingen, San Diego, CA, USA). The remaining CD19+ and CD19- fractions were immediately lysed in 1 mL of the Trizol reagent (Invitrogen) and frozen at -80oC.
Extracted molecule total RNA
Extraction protocol Trizol cell lysates were mixed with chloroform (1/5 of lysate volume), vortexed for one minute and centrifuged in a table-top centrifuge at 13,000 rpm for 15 min at 4ºC. The aqueous phase was used for RNA isolation. The aqueous phase was mixed with equal volume of 70% ethanol and immediately loaded onto RNeasy mini columns (Qiagen, Valencia, CA, USA), with subsequent steps performed as per the manufacturer’s protocol. The RNA quality was estimated on a 2100 Bioanalyzer, RNA 6000 Nano Chips (Agilent, Santa Clara, CA, USA). Samples with RNA integrity number (RIN) of 8.0 and above were used for further analysis.
Label Cy3
Label protocol Biotinylated cRNA were prepared with the Illumina TotalPrep RNA amplification kit (Ambion Inc., Austin, TX) per the manufacturer's instuctions.
 
Hybridization protocol Amplified biotinylated cRNA (1.5 ug) were hybridized to Mouse Ref-6_v1 Sentrix BeadChips. In all steps, care was exercised to avoid batch effects. Samples were hybridized to the microarrays at 55ºC for 16-17 hours. Microarrays were washed to remove non-specifically bound cRNA, stained with 1 mg/mL Streptavidin-Cy3 (GE Healthcare, Piscataway, NJ, USA) and dried.
Scan protocol Microarrays were scanned in BeadStation 500 scanner. Image acquisition and initial image analysis were done with Illumina BeadScan and BeadStudio applications.
Description 12-wt
Data processing Raw data from the Illumina BeadChips was corrected for background and quantile normalized using BeadExplorer (version 1.5.0), a Bioconductor module developed for quality control, normalization, annotation and exploration of Illumina BeadChip data. Average detection value was calculated for each transcript, and the transcripts with average detection values below 0.99 were excluded from further analysis.

Non-normalized data is available in the 'GSE18447_non-normalized_data.txt' file, which is linked to the Series GSE18447 record as a supplementary file.
 
Submission date Oct 06, 2009
Last update date Oct 06, 2009
Contact name Alexander Pemov
Organization name National Institutes of Health
Street address 49 Convent Drive
City Bethesda
State/province MD
ZIP/Postal code 20814
Country USA
 
Platform ID GPL6333
Series (2)
GSE18447 Genome-wide transcriptional profiling of Nf1-haploinsufficiency in mouse
GSE18448 Genome-wide transcriptional profiling of Nf1-haploinsufficiency

Data table header descriptions
ID_REF
VALUE Quantile-normalized signal intensity
DETECTION

Data table
ID_REF VALUE DETECTION
GI_38088637-S 158.0102076 0.99257426
GI_38074118-S 228.7929417 1
scl42124.1_5-S 159.8998754 0.99319307
scl011813.3_68-S 151.8048354 0.99071782
scl0002405.1_260-S 169.42725 0.99628713
scl27219.2.1_88-S 159.1351331 0.99257426
GI_20895633-S 182.8419142 0.99876238
GI_38075382-S 163.5970681 0.99381188
scl0001724.1_23-S 168.7288581 0.99628713
scl40977.18.1_213-S 163.5970681 0.99381188
scl069236.1_275-S 161.8515799 0.99381188
scl23047.4.213_30-S 139.780897 0.96720297
scl000214.1_15-S 153.3259995 0.99133663
scl30798.9_425-S 158.0102076 0.99257426
scl36309.11_147-S 150.0065262 0.99071782
scl00238247.1_16-S 189.1138068 0.99876238
scl074650.1_112-S 157.2538594 0.99257426
scl33279.12.1_23-S 152.5116202 0.99133663
GI_38348461-S 157.6777756 0.99257426
scl0011789.2_94-S 219.5940876 1

Total number of rows: 12860

Table truncated, full table size 447 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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