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| Status |
Public on Feb 06, 2013 |
| Title |
macrophages_treated_with_LPS+PGE_1hr_rep_2 |
| Sample type |
RNA |
| |
|
| Source name |
primary bone marrow derived mouse macrophages, treated with LPS and PGE, harvested after 1 hour of treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6 wild type mouse
cell type: primary macrophages
tissue: bone marrow
|
| Treatment protocol |
BMDMs were either left untreated or stimulated with 100 ng/ml LPS (#L6529; Sigma) or a combination of 100 ng/ml LPS and 10 microM prostaglandin E for 1 hour
|
| Growth protocol |
To obtain primary bone marrow derived mouse macrophages (BMDMs), bone marrow cells were maintained on bacterial grade plates for 1 week in DMEM supplemented with 10% heat inactivated FBS (Biosera), 2 mM L-glutamine, 100 units/ml penicillin G, 100mg/ml streptomycin, 0.25 mg/ml amphotericin (Invitrogen) and 5 ng/ml mCSF (R&D systems). Adherent cells were then re-plated on tissue culture grade plates in fresh media and used 24 hours after re-plating. RAW264.7 cells were cultured in DMEM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin G and 100mg/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was carried out by homogenisation of BMDMs in Trizol followed by phenol chloroform extraction.
|
| Label |
biotin
|
| Label protocol |
250 ng of total RNA was used from each sample. The total RNAs were processed with Ambion WT Expression kit (P/N 4411973) and Affymetrix Genechip® WT Terminal Labelling and Controls Kit (P/N 901524), according to manufactures protocols. RNA concentration was checked with Nanodrop ND-2000 and Agilent Bioanalyzer electrophoresis station was used for quality control.
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| |
|
| Hybridization protocol |
Samples were hybridized to Affymetrix Mouse Gene 1.1 ST array plate using manufacturer's protocols for using the GeneTitan® Hybridization, Wash and Stain Kit for WT Array Plates (P/N 901622)
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| Scan protocol |
GeneTitan® Instrument was used to hybridize, wash, stain and scan the arrays. Affymetrix GeneChip® Command Console® (AGCC) 3.1 was used to control GeneTitan® hybridization process and in summarizing probe cell intensity data (.CEL file generation). The hybridization quality was checked with AGCC and Expression Console 1.1.
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| Data processing |
The data analysis was carried out using Affymetrix Power Tools (APT), R (version 2.13.1) – Bioconductor and Partek GS 6.5 (version 6.11.0321) software. For quality control and probesets annotations, the latest annotations files (Release 32, dated 23-06-2011) downloaded from Affymetrix were used. Using APT, The probe level expression data were summarised to transcript cluster level, filtered to include DABG (detected above background) and normalised using two different normalisation methods – RMA and Plier-GCBA (GC background-adjusted). The dataset quality was analysed from spike in probesets, principal component analysis, hierarchical clustering analysis and by generating various quality control plots such as mean raw signal intensity plots, mean absolute deviation (MAD) of the residuals plots and probe cell intensity plots. The processed dataset submitted here (matrix table) shows quality control passed, DABG (detected above background) filtered data summarised to transcript cluster level and normalised using RMA normalisation method using Affymetrix Power Tools.
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| |
|
| Submission date |
Oct 25, 2012 |
| Last update date |
Feb 06, 2013 |
| Contact name |
Manikhandan A V Mudaliar |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Glasgow
|
| Department |
College of Medical, Veterinary and Life Sciences
|
| Lab |
Institute of Infection, Immunity and Inflammation
|
| Street address |
Room B528, Sir Graeme Davies Building, 120 University Place
|
| City |
Glasgow |
| State/province |
Scotland |
| ZIP/Postal code |
G12 8TA |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11533 |
| Series (1) |
| GSE41833 |
Expression profiles of primary bone marrow derived mouse macrophages – untreated and treated with LPS or LPS+PGE |
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