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Sample GSM691441 Query DataSets for GSM691441
Status Public on May 22, 2011
Title macrophage_TrifKO_E.coli_6h_rep2
Sample type RNA
 
Source name bone marrow derived macrophages from Trif knockout mice, stimulated with E.coli for 6hrs
Organism Mus musculus
Characteristics strain: C57BL6/J
gender: Female
age: 5-6 weeks
genotype/variation: Trf1 knock out
cell type: bone marrow macrophage (BMM)
protocol: E.coli stimulation
time: 6h
Treatment protocol The cultured bone marrow derived macrophages (BMMs) were detached and re-plated 4 hours prior to the experiment. At the start of the experiment, BMMs were incubated with viable E. coli for the 1, 3 or 6 hours. Bacteria were used at a multiplicity of infection (MOI) of 20. All experiments were carried out in antibiotic free 'complete medium'. One hour after addition of viable bacteria, penicillin (100μg/ml) and streptomycin (100μg/ml) were added to the media in order to kill any remaining extracellular bacteria. For transcriptional analysis three independent experiments were performed. We compared this approach to washing the cells and replacing the antibiotic free medium with penicillin/streptomycin containing medium after one hour and found no differences with regards to the cellular responses measured.
Growth protocol Murine macrophages, derived from the bone marrow of either C57BL/6J, or Trif-/- mice, were cultured in RPMI 1640 supplemented with macrophage colony-stimulating factor (M-CSF) and 10% FBS, plus 100μg/ml penicillin, 100μg/ml streptomycin, 10mM HEPES and 1nM sodium pyruvate (all obtained from Sigma-Aldrich).
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from the BMMs using the RNeasy kit (QIAGEN). RNA from RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) with 6000 Nano Chips. RNA was judged as suitable only if samples showed intact bands of 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RNA integrity number (RIN) above 8.0.
Label biotin
Label protocol The Affymetrix GeneChip WT Sense Target Labeling and Control Reagents kit (P/N 900652) was used for the preparation of labelled cDNA from 100ng of total RNA without rRNA reduction. A detailed description can be found in the User Manual, Chapter 3 (P/N 701880, revision 5).
 
Hybridization protocol The Affymetrix GeneChip Mouse Gene 1.1 ST 24-array plate consists of 24 single Gene 1.1 ST arrays arranged into the standard 96 well plate format. Array hybridization, washing and scanning were performed on a GeneTitan Instrument according to the manufacturer’s recommendations.
Scan protocol Arrays were scanned on an Affymetrix GeneTitan instrument.
Description KO_Ecoli6h_2
G004_H07_16_114-8_KO_Ecoli6h_2.CEL
Data processing Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.14.0).
 
Submission date Mar 15, 2011
Last update date May 22, 2011
Contact name Guido Hooiveld
E-mail(s) guido.hooiveld@wur.nl
Organization name Wageningen University
Department Div. Human Nutrition & Health
Lab Nutrition, Metabolism & Genomics Group
Street address HELIX, Stippeneng 4
City Wageningen
ZIP/Postal code NL-6708WE
Country Netherlands
 
Platform ID GPL11533
Series (1)
GSE27960 Sensing prokaryotic mRNA signifies microbial viability and promotes immunity

Data table header descriptions
ID_REF
VALUE RMA signal (as log2)

Data table
ID_REF VALUE
10338001 11.24728026
10338002 5.084506074
10338003 9.351239055
10338004 8.691580272
10338005 2.490295951
10338006 2.686116522
10338007 2.96834709
10338008 3.321568633
10338009 7.063230592
10338010 2.502505527
10338011 4.512567987
10338012 2.585727079
10338013 2.263037231
10338014 2.403179593
10338015 2.315593069
10338016 6.053891675
10338017 12.49632605
10338018 5.569494702
10338019 4.051596237
10338020 6.879075119

Total number of rows: 35556

Table truncated, full table size 725 Kbytes.




Supplementary file Size Download File type/resource
GSM691441.CEL.gz 4.7 Mb (ftp)(http) CEL
Processed data included within Sample table

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