strain: C57BL/6 genotype/variation: knockout (HDAC3 -/-) cell type: Primary bone marrow-derived macrophages (BMDM) cells (7th day of differentiation) treatment: LPS (100 ng/ml) for 4hrs
Treatment protocol
Macrophages were stimulated with lipopolysaccharide (LPS, 100 ng/ml)
Growth protocol
Bone marrow cells isolated from C57BL/6 mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% β-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days.
Extracted molecule
total RNA
Extraction protocol
RNeasy from Qiagen with DNAse I treatment
Label
biotin
Label protocol
Biotin-labeled cDNA targets were synthesized starting from 150 ng of totRNA. Double stranded cDNA synthesis and related cRNA amplification was performed with Ambion® WT Expression Kit (Ambion, Austin, TX). cRNA was reverse transcribed to cDNA fragmented and terminal labeled with Affymetrix GeneChip® WT Teminal Labeling Kit (Affymetrix, Santa Clara, CA). All steps of the protocol were performed according to manufacturer’s specifications. Efficacy of fragmentation procedure was checked on Agilent Bioanalyzer 2100.
Hybridization protocol
GeneChip Hybridization, Wash and Stain Kit (Affymetrix cat #900720) is used to prepare the Hybridization Cocktail. The kit contains mix for target dilution, DMSO at a final concentration of 7% and pre-mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and Cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively. These control oligos serve as positive controls for hybridization. Biotin-labeled sense targets were diluted in hybridization buffer at a concentration of 25 ng/ul and denatured at 99 C for 5 minutes. Hybridization cocktails were incubated at 45 °C for 5 minutes and centrifuged at maximum speed for 1 minute prior to loading into the GeneChip array cartridge. Hybridizations were performed for 16 h at 45C in a rotisserie oven at 60 RPM. Upon hybridization, GeneChip cartridges were washed and stained with GeneChip Hybridization, Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450_0007 standard protocol. The cartridge is then loaded into the GeneChip Scanner 3000 7G.
Scan protocol
GeneChip arrays are scanned using default parameters. Affymetrix GeneChip Command Console software (AGCC) was used to acquire GeneChip images and generate .DAT and .CEL files.
Description
KO_LPS_4h_rep2
Data processing
CELs files were imported into R, RMA normalized and annotated using the Affy package.
