|
| Status |
Public on May 21, 2015 |
| Title |
LPS 24hr B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LPS 24hr B
|
| Organism |
Mus musculus |
| Characteristics |
treatment: LPS
timepoint: 24hr
donor: B
|
| Treatment protocol |
Day 5 BMDCs were stimulated with 100ng/ml LPS (Sigma) for 0, 1, 6, or 24 hours. DCs were then collected by repeated pipetting.
|
| Growth protocol |
Bone marrow was harvested from mouse femur and tibia. B and T cells were depleted using biotinylated antibody cocktail and streptavidin magnetic beads. Non-depleted cells were cultured in RPMI supplemented with 10ng/ml GM-CSF and 5ng/ml IL-4 recombinant cytokines for 5 days. One half culture medium was replaced with fresh growth medium every other day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from collected cells using RNeasy Plus Kit (Qiagen) following manufacturers protocol.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was converted to double-stranded cDNA and then into Cy-dye labeled cRNA using Agilent’s Low RNA Input Fluorescent Linear Amplification Kit. 750 ng of the labeled cRNA was fragmented and hybridized against Cy3-labeled Universal mouse reference (Stratagene, La Jolla, CA).
|
| |
|
| Channel 2 |
| Source name |
Universal mouse reference (UMR) from Stratagene
|
| Organism |
Mus musculus |
| Characteristics |
sample type: pooled mouse reference RNA
|
| Treatment protocol |
Day 5 BMDCs were stimulated with 100ng/ml LPS (Sigma) for 0, 1, 6, or 24 hours. DCs were then collected by repeated pipetting.
|
| Growth protocol |
Bone marrow was harvested from mouse femur and tibia. B and T cells were depleted using biotinylated antibody cocktail and streptavidin magnetic beads. Non-depleted cells were cultured in RPMI supplemented with 10ng/ml GM-CSF and 5ng/ml IL-4 recombinant cytokines for 5 days. One half culture medium was replaced with fresh growth medium every other day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from collected cells using RNeasy Plus Kit (Qiagen) following manufacturers protocol.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was converted to double-stranded cDNA and then into Cy-dye labeled cRNA using Agilent’s Low RNA Input Fluorescent Linear Amplification Kit. 750 ng of the labeled cRNA was fragmented and hybridized against Cy3-labeled Universal mouse reference (Stratagene, La Jolla, CA).
|
| |
|
| |
| Hybridization protocol |
Agilent In Situ Hybridization Kit Plus was used where Cy-dye labeled control and test samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, the arrays were washed twice, dried and then scanned.
|
| Scan protocol |
Scanned on an Agilent scanner, images were processed using Agilent Feature Extraction software version 10.7.
|
| Description |
Biological replicate 2 of 3: BMDCs stimulated with LPS for 24hr
|
| Data processing |
Raw intensity data were background subtracted using the normal + exponential method from the limma Bioconductor package. The test/reference ratio was log2 transformed and normalized using loess normalization for each array. Loess normalized log2 ratios were then subjected to quantiles normalization across all arrays in the dataset.
|
| |
|
| Submission date |
May 20, 2015 |
| Last update date |
May 21, 2015 |
| Contact name |
Jason A Hackney |
| E-mail(s) |
hackney.jason@gene.com
|
| Organization name |
Genentech, Inc.
|
| Department |
Department of Bioinformatics and Computational Biology
|
| Street address |
1 DNA Way
|
| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE69076 |
Identification of LPS-responsive genes in bone marrow-derived dendritic cells |
|
