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Sample GSM691427 Query DataSets for GSM691427
Status Public on May 22, 2011
Title macrophage_wildtype_E.coli_3h_rep3
Sample type RNA
Source name bone marrow derived macrophages from WT (C57BL6/J) mice, stimulated with E.coli for 3hrs
Organism Mus musculus
Characteristics strain: C57BL6/J
gender: Female
age: 5-6 weeks
genotype/variation: wild type
cell type: bone marrow macrophage (BMM)
protocol: E.coli stimulation
time: 3h
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 WT_Ecoli3h_3
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
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
VALUE RMA signal (as log2)

Data table
10338001 11.39214287
10338002 5.214729563
10338003 9.662254378
10338004 8.798376694
10338005 2.439489116
10338006 2.594854039
10338007 2.920107623
10338008 3.301296209
10338009 7.304372094
10338010 2.409592898
10338011 4.576824836
10338012 2.508991139
10338013 2.232542113
10338014 2.338531605
10338015 2.246605173
10338016 6.29597734
10338017 12.44097888
10338018 5.695488651
10338019 4.091071458
10338020 7.129324323

Total number of rows: 35556

Table truncated, full table size 725 Kbytes.

Supplementary file Size Download File type/resource
GSM691427.CEL.gz 4.6 Mb (ftp)(http) CEL
Processed data included within Sample table

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