strain background: C57BL genotype/variation: C57BL/10NAGCSnAi-(KO) Rag2 age: 6 weeks tissue: bone marrow cell type: dendritic cells (DCs) stimulated with: 1ug/ml Lipopolysaccharide for 18 h
Treatment protocol
DCs were incubated with 1 ug/ml of Hematoporphyrin (HP; 8,13-Bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H, 23H-porphine-2,18-dipropionic acid, Sigma Aldrich, Saint Louis, MO, USA) for 1 h, washed twice with complete IMDM medium without phenol red, illuminated by a home built light emitting diode (LED), which has wide excitation wavelength ranging from 450 to 600 with peak wavelength at 515 nm at the intensity of 3.1 mW cm-2, equipped with a UV cutoff filter for 3 min, and then cultured for 18 h to prepare total RNA. DCs were also stimulated with 1 ug/ml Lipopolysaccharide (LPS; Sigma Aldrich) for 18 h.
Growth protocol
DCs were generated from the bone marrow of six-week-old Rag2 KO mouse. The Bone marrow cells were flushed out of the femurs and tibias with Iscove`s modified eagle medium (IMDM, Gibco Invitrogen, Grand Island, NY, USA) medium. DCs were culture in IMDM supplemented with 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng/ml, PeproTech, Rocky Hill, NJ, USA), recombinant mouse IL-4 (1.5 ng/ml, PeproTech), penicillin (100 units/ml, Gibco invitrogen), streptomycin (0.1 mg/ml, Gibco Invitrogen), gentamicin (0.05 mg/ml, Gibco Invitrogen), L-Glutamine (2 mM, Gibco Invitrogen), and 2-mercaptoethanol (50 nM, Gibco Invitrogen). Half of the medium was replaced every other day with an equal volume of complete IMDM medium for 6 d.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, USA), purified using RNeasy columns (Qiagen, Valencia, USA) according to the manufacturers’ protocol.
Label
biotin
Label protocol
Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cRNA according to the manufacturer’s instructions.
Hybridization protocol
750 ng of labeled cRNA samples were hybridized to each mouse-8 expression bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina, Inc., San Diego, USA). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual.
Scan protocol
Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions.
Description
replicate-2
Data processing
Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4)). Array data were filtered by detection p-value < 0.05 (similar to signal to noise) in at least 50% samples (we applied a filtering criterion for data analysis; higher signal value was required to obtain a detection p-value < 0.05). Selected probe signal value was transformed by logarithm and normalized by quantile method. The comparative analysis between test group and control group was carried out using LPE test and fold-change. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm.
