|
| Status |
Public on Dec 13, 2007 |
| Title |
pDC CpG 1h, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
pDC cultured in medium with CpG for 1h
|
| Organism |
Mus musculus |
| Characteristics |
pDC isolated from spleen of Flt-3L-treated DPEGFPxRag1-/- mice
|
| Treatment protocol |
pDC were sorted from spleens of Flt-3L-treated DPExRag1-/- mice and cultured at 1x106/ml for 1h with 5 µg/ml CpG 1826 (MWG) in complete media (RPMI supplemented with 10% heat-inactivated FBS (Valley Biomedical, Inc.), Penicillin/Streptomycin (Invitrogen), 10mM HEPES (Invitrogen), 2mM L-Glutamine (Invitrogen), 10mM pyruvate (Invitrogen), 50µM 2-mercaptoethanol (Sigma).
|
| Growth protocol |
DPEGFPxRag1-/- were injected 12-14 days prior to sort with 2x10^6 B16F10-Flt-3L cells, which leads to expansion of immature pDCs in spleens of mice
|
| Extracted molecule |
total RNA |
| Extraction protocol |
isolation of total RNA was performed using the Rneasy Micro Kit according to manufacturer's instructions (Qiagen)
|
| Label |
biotin
|
| Label protocol |
5ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporates the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP using the Enzo High-Yield amplification kit.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99oC for 5 min and hybridized for 16 h at 45oC to GeneChip microarrays. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
The GeneChip 3000 scanner was used to collect fluorescence signal after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
| Description |
Gene expression data from activated plasmacytoid dendritic cells with CPG for 1h
|
| Data processing |
Data were processed using Affymetrix GCOS v1.4 software and probe intensity files (.cel files) were further analyzed using the GeneSpring v7.0 software (Silicon Genetics). GCRMA algorithm was used to summerize and normalize data.
|
| |
|
| Submission date |
May 17, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Amaya I Wolf |
| E-mail(s) |
aiwolf@wistar.org
|
| Organization name |
Wistar Institute
|
| Street address |
3601 Spruce street
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE7831 |
Expression data from immature pDC and pDC activated with CpG 1826 and influenza virus PR8 |
|
| Relations |
| Reanalyzed by |
GSE119085 |
