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Sample GSM525096 Query DataSets for GSM525096
Status Public on Mar 23, 2010
Title DN3 rep2 (4459290113_D)
Sample type RNA
 
Source name mice lymphocytes
Organism Mus musculus
Characteristics strain: C57B7
cell type: DN3 (parental T cells)
Treatment protocol Thymocytes or splenocytes from Cre-ERT2; Bcl11bflox/flox mice were cultured in T cell medium with 1 μM 4-OHT (Sigma) at 370 C for 48 hrs. After this time, cells were washed and resuspended with fresh media. T cell media: RPMI-1640, 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine, 5 ng/ml muFlt-3-L, 5 ng/ml huIL-7.
Growth protocol After 4-hydroxytamoxifen (OHT) treatment, DN3 thymocytes from Bcl11bCKO/CKO and Bcl11bCKO/+ were sorted by FACS and co-cultured with OP9-DL1 in T cell culture media (3,000 cells per well in 24-well plates) with 100 ng/ml huIL-2, respectively. Every three days, cells were collected by vigorous pipetting, filtered through cell strainers and transferred to new tissue culture plates pre-seeded with fresh OP9-DL1 stromal cells. Fourteen days after OHT treatment, cells were collected for RNA extraction. For ex vivo expansion, splenic ITNK cells were enriched using the NK Isolation Kit (Miltenyi) and cultured for 6-9 days at 1 x 106 cells/ml in RPMI 1640 medium containing 10% FCS/50 µM 2-mercaptoethanol/2.0 mM L-glutamine and 1000 U/ml hIL-2 (Chiron).
Extracted molecule total RNA
Extraction protocol RNA from primary cells was extracted using the RNAqueous-Micro RNA isolation kit according to the manufacture protocol (Ambion). Briefly, cells were counted and pelleted by centrifuge at 4,000 G for 5 min. Remove supernatant thoroughly by aspiration. Resuspend cell pellet by vortexing vigorously in at least 100 ul Lysis Solution. Then 50 ul of 100% ethanol was added and mixed with lysate thoroughly. Next load the lysate/ethanol mixture onto a Micro Filter Cartridge Assembly and centrifuge for 10 sec at 16,000 G. Then wash the filter with 180 ul of Wash Solution 1 once and 180 ul of Wash Solution 2/3 twice. Discard the flow-through and centrifuge the filter for 1 min at 16,000 G. Elute the RNA into a Micro Elution Tube with 2X5 ul preheated Elution Solution. To eliminate contaminated DNA from extracted RNA, 1/10th volume of 10X DNase I Buffer and 1 ul of DNase I were added into the RNA sample and mixed gently but thoroughly. After incubation at 20 min at 370C, the DNase reaction was mixed with 2 ul DNase Inactivation Reagent and left at room temperature for 2 min. Finally, pellet the DNase Inactivation Reagent and transfer the RNA to a fresh tube. For RT-PCR or qRT-PCR experiments, RNA quality and quantity was verified using a Nanodrop ND-100 Spectrophotometer (Thermo Scientific). For gene expression array experiments, RNA quality and quantity was determined using 2100 Bioanalyzer platform (Aglient Technologies).
Label biotin
Label protocol Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
 
Hybridization protocol Standard Illumina hybridization protocol
Scan protocol Standard Illumina scanning protocol
Description replicate 2
Data processing The data were normalized using quantile normalization with dChip
 
Submission date Mar 23, 2010
Last update date Mar 23, 2010
Contact name Jack Chen
Organization name NIH
Street address SAIC-Frederick Miller Dr
City Frederick
State/province MD
ZIP/Postal code 21702
Country USA
 
Platform ID GPL6887
Series (1)
GSE21016 Genome-wide analysis of gene expression profiles in ITNK

Data table header descriptions
ID_REF
VALUE quantile normalized log2 signal intensity
Detection-4459290113_D

Data table
ID_REF VALUE Detection-4459290113_D
ILMN_1212602 6.1 0.57692
ILMN_1212603 6.07 0.5203
ILMN_1212605 8.46 1
ILMN_1212607 6.04 0.44658
ILMN_1212612 6.24 0.87821
ILMN_1212614 5.89 0.11645
ILMN_1212619 6.15 0.70833
ILMN_1212623 6.3 0.94017
ILMN_1212625 8.18 1
ILMN_1212626 6.22 0.85791
ILMN_1212628 6.19 0.79808
ILMN_1212632 6.07 0.54594
ILMN_1212633 6.15 0.68803
ILMN_1212636 10.31 1
ILMN_1212637 6.92 1
ILMN_1212638 12.22 1
ILMN_1212639 6.24 0.88568
ILMN_1212644 6.19 0.80449
ILMN_1212645 6.15 0.70513
ILMN_1212646 8.21 1

Total number of rows: 45281

Table truncated, full table size 1078 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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