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Sample GSM691424 Query DataSets for GSM691424
Status Public on May 22, 2011
Title macrophage_wildtype_E.coli_1h_rep3
Sample type RNA
 
Source name bone marrow derived macrophages from WT (C57BL6/J) mice, stimulated with E.coli for 1hr
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: 1h
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_Ecoli1h_3
G004_B09_18_117-2_WT_Ecoli1h_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 11.33026021
10338002 5.042245666
10338003 9.339924296
10338004 8.475648963
10338005 2.403963659
10338006 2.612150714
10338007 2.865743382
10338008 3.219404465
10338009 6.934090637
10338010 2.424708948
10338011 4.374263334
10338012 2.49989929
10338013 2.222893521
10338014 2.329602562
10338015 2.22823785
10338016 5.966865127
10338017 12.40232995
10338018 5.435248471
10338019 4.012099963
10338020 6.731439338

Total number of rows: 35556

Table truncated, full table size 725 Kbytes.




Supplementary file Size Download File type/resource
GSM691424.CEL.gz 4.7 Mb (ftp)(http) CEL
Processed data included within Sample table

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