mouse-strain: Balb-C sex: Male age: 8-9 weeks cell type: Bone Marrow Derived Macrophage
Treatment protocol
On day 7 of growth BMDM cells were treated with 10 U/ml recombinant mouse interferon-beta (Ifn-β) (PBL Interferon Source, New Jersey, USA) and harvested 1, 2, 4, 8 and 24 h following treatment or collected pre-treatment (0 h). Duplicate biological replicates for each time-point were processed for hybridization to the Mouse Exon 1.0 ST Arrays.
Growth protocol
Bone marrow derived macrophages (BMDM) were prepared from femurs of 8-9 week old male Balb-C mice. RPMI 1640 medium (Sigma-Aldrich, Gillingham, UK) supplemented with 10% heat inactivated foetal bovine serum (FBS) (Sigma-Aldrich Gillingham, UK), 25 U/ml penicillin (Invitrogen, Paisley, UK), 25 µg/ml streptomycin (Invitrogen, Paisley, UK), and 2 mM L-glutamine (Invitrogen) (complete medium) was used for culture of the BMDM. Briefly, bone marrow cells were cultured for 6 days in complete medium in the presence of 10,000 U/ml CSF1 (colony stimulating factor-1) on 10 cm square bacteriological plastic plates, with a re-supplement of CSF1 on day 5. On day 6 cells were harvested, counted, re-suspended in complete medium with 10,000 U/ml CSF1 and plated out onto 6-well tissue culture plates at a density of 1 million cells per well and cultured for a further 24 h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was harvested from the cells using an RNeasy Mini kit (Qiagen, Crawley, UK) according to manufacturer's instructions. RNA was quantified and quality controlled using a NanoDrop spectrophotometer (NanoDrop Technologies, Delaware, USA) and BioAnalyser 2100 (Agilent, California, USA). Replicate 300 ng samples of total RNA derived from two separate wells per time point were labeled using the Affymetrix whole transcript labeling protocol.
Label
biotin
Label protocol
Single-stranded cDNA was generated from the amplified cRNA with the WT cDNA Synthesis Kit (Affymetrix) and then fragmented and labeled with the WT Terminal Labeling Kit (Affymetrix).
Hybridization protocol
Samples were hybridized with GeneChip Mouse Exon 1.0 ST Arrays (Affymetrix) and scanned at the Wellcome Trust Sanger Institute.
Scan protocol
Array scanning was performed according to the manufacturer's instruction (Affymetrix)
Description
RMA expression value derived from Expression Console software; Core-Gene-Level analysis
Data processing
Raw data was normalized using the RMA package within the Affymetrix Expression Console software and annotated. Transcripts which might be considered to be differentially expressed at each time point compared to 0 h were identified using the Empirical Bayes function within the Bioconductor package of the R statistical program using a 1.5 fold cut-off and a p-value of 0.05. According to this analysis 2,300 transcripts were considered to be differentially expressed. Data corresponding to these transcripts was then loaded into the network visualization tool BioLayout Express3D using a Pearson correlation cut-off of 0.9 to filter edges. The the graph-based clustering algorithm MCL and an explorative network based approach was applied to analyse transcriptional changes over time.
