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Sample GSM363716 Query DataSets for GSM363716
Status Public on Aug 01, 2009
Title Traf1_IFN-_A21A TF_RNAi-EXON_180407
Sample type RNA
 
Source name BMDMs, IFNg stimulation, siRNA treatment
Organism Mus musculus
Characteristics Balb/c male mice age 10-12 weeks. Bone marrow (containing primary mouse monocytes) was flushed from femurs and differentiated for 7 days in DMEM-F12/10% FCS/10% L929 medium (releasing CSF-1). Cells grown in 24-well plates at a seeding density of 5x105 cells/well. Media changes on days 3 and 5.
Treatment protocol Transfection of siRNA and IFNγ treatment Differentiated primary macrophages were subject to siRNAs SMARTpools (Thermo Fisher Inc) designed to each mRNA target (4 siRNAs per pool). siRNAs were purchased at a 5 nmol scale and redissolved in 1x siRNA buffer (Thermo Fisher Inc) to a final concentration of 1 µM. To transfect at a final concentration of 20 nM, 1 µl of siRNA SMARTpool was used with 49 µl of Optimem (Invitrogen, CA, USA) solution while 2 µl of Lipofectamine 2000 (L2K, Invitrogen) was mixed with 48 µl Optimem. Following incubation for 5 min, the siRNA mix was added to the L2K mix and incubated for a further 30 min, after which 400 µl of DMEM-F12/10% FCS/L929 medium lacking antibiotics was added to the siRNA:L2K complexes. Growth medium was removed and cells were washed 1x in PBS before 500 µl of the siRNA:L2K liposomes were added. Cells were then incubated for a further 24 h (37ºC, 5% CO2). For IFNγ treatments, growth medium was replaced with medium containing 10 U/ml recombinant mouse IFNγ. Cells were incubated for 24 h prior to harvesting of total RNA.
Growth protocol Balb/c male mice age 10-12 weeks. Bone marrow (containing primary mouse monocytes) was flushed from femurs and differentiated for 7 days in DMEM-F12/10% FCS/10% L929 medium (releasing CSF-1). Cells grown in 24-well plates at a seeding density of 5x105 cells/well. Media changes on days 3 and 5.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using an RNeasy Plus kit (Qiagen) according to manufacturer's instructions. RNA was quantified and quality controlled using a NanoDrop spectrophotometer (NanoDrop Technologies) and BioAnalyser 2100 (Agilent). They were then washed and scanned according to manufacturer's recommendations
Label biotin
Label protocol Microarray target labelling: 150 ng of total RNA was processed using the Exon array target labelling kit (Affymetrix, Santa Clara, US) according to manufacturer’s instructions for small sample labelling without the use of the RiboMinus step.
 
Hybridization protocol Hybridization according to Affymetrix Mouse Exon Array 1.0ST protocol (Affymetrix, Santa Clara, US)
Scan protocol Affymetrix Genechip 7G ScanMachinePrt1
Description Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays.
Data processing RMA normalization using "core" set of probes
 
Submission date Jan 23, 2009
Last update date May 26, 2009
Contact name Paul A Lacaze
Organization name University of Edinburgh
Department School of Medicine
Lab Division of Pathway Medicine
Street address 49 Little France Crescent
City Edinburgh
ZIP/Postal code EH16 4SB
Country United Kingdom
 
Platform ID GPL6096
Series (1)
GSE14534 Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages

Data table header descriptions
ID_REF
VALUE Quantification

Data table
ID_REF VALUE
6848511 8.71689
6864895 8.56056
6766590 1.95979
6914045 1.6716
6963197 3.93651
6766588 1.68487
6995964 9.4804
6766587 2.91914
6815739 3.15676
6766586 4.73775
7012346 2.45014
6766585 4.19117
6848505 5.96408
6766584 1.62905
6848504 8.50921
6979576 2.85924
6995960 3.02104
6766583 2.63949
6963191 3.70925
6979575 4.1196

Total number of rows: 23238

Table truncated, full table size 360 Kbytes.




Supplementary file Size Download File type/resource
GSM363716.CEL.gz 20.7 Mb (ftp)(http) CEL
GSM363716.chp.gz 165.0 Kb (ftp)(http) CHP
Processed data included within Sample table
Processed data provided as supplementary file

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