|
| Status |
Public on Aug 31, 2015 |
| Title |
Pooled CD11b+Gr1+ splenocytes from 3 healthy L2-Cre;p120+/f mice, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
control pool 3
|
| Organism |
Mus musculus |
| Characteristics |
age: E11.5
tissue: spleen
genotype: Shh -/-
strain: mixed C57BL/6x129 background
|
| Treatment protocol |
not applicable
|
| Growth protocol |
spleens were extracted from experimental and control mice
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Splenocytes were isolated by FACS sorting (CD11b+Gr1+ cells were collected) directly into the QIAzol lysis reagent (QIAGEN), and total RNA extraction was carried out according to the manufacturer's protocol. Quality control check of the total RNA samples was performed using Agilent Bioanalyzer and Nanodrop spectrophotometry. All protocols were conducted as described in the Ambion WT Expression Manual and the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 250ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA.
|
| Label |
biotin
|
| Label protocol |
The cDNA were fragmented, assessed by Bioanalyzer, and biotinylated using terminal transferase end labeling.
|
| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix hybridization kit materials. 5.5 micrograms of labeled cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Mouse Exon 1.0 ST GeneChips (Affymetrix Inc., Santa Clara CA) using the GeneChip Hybridization oven 645. 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 |
A GeneChip 3000 7G scanner was used to collect fluorescence signal. Affymetrix Command Console and Expression Console were used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters.
|
| Description |
Con_03
Forelimbs from Shh mutants 964 and 965
|
| Data processing |
All samples were carried out by doing background subtraction, normalization and summarizing probe sets from Affymetrix expression microarrays using Affymetrix Power tools (APT). The data were processed with RMA (Robust Multichip Average) and Probe Logarithmic Intensity Error (PLIER) Estimation methods in APT.
MoEx-1_0-st-v1.r2.pgf
MoEx-1_0-st-v1.r2.dt1.mm9.core.mps
|
| |
|
| Submission date |
Aug 04, 2015 |
| Last update date |
Sep 21, 2015 |
| Contact name |
Anil K. Rustgi |
| Organization name |
University of Pennsylvania
|
| Department |
Gastroenterology
|
| Lab |
Rustgi Lab
|
| Street address |
421 Curie Boulevard
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (2) |
| GSE71706 |
Expression data from CD11b+Gr1+ splenocytes from tumor-bearing L2-Cre;p120f/f mice, compared to controls |
| GSE71707 |
Myeloid derived suppressor cells from tumor-bearing L2-Cre;p120f/f mice |
|
