|
| Status |
Public on Oct 29, 2013 |
| Title |
male WT, replicate C |
| Sample type |
RNA |
| |
|
| Source name |
TGC-induced peritoneal macrophage
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
genotype: WT
cell type: macrophage
genetic background: C57BL/6 background, p38 floxed mice (Mapk14tm1.2Otsu) crossed with Lysm-Cre mice
|
| Treatment protocol |
all samples were stimulated for 4 hrs with heat killed mycobacterium tuberculosis
|
| Growth protocol |
macrophages were isolated on D4 post thioglycolate injection, adhered to plates overnight
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the RNEasy kit (Qiagen) according to manufacturer’s instructions. Microarray was performed on 3 biological replicates for each condition (e.g. female KO). To create each replicate, equal amounts of RNA from 2-3 different mice were pooled. An RNA input of 25ng was used to generate cDNA through the First Strand and Second Strand synthesis reactions of the Ovation® Pico WTA System V2 from NuGEN. The cDNA samples were then purified using an Agencourt® RNAClean® XP magnetic bead protocol. Following purification, samples were amplified using SPIA reagents from the Ovation® Pico WTA System V2 from NuGEN. A final cDNA purification is performed using an Agencourt® RNAClean® XP magnetic bead protocol.
|
| Label |
biotin
|
| Label protocol |
Approximately 4ug of cDNA generated using the Ovation® Pico WTA System V2 was fragmented and labeled using the Encore® Biotin Module from NuGEN. Efficiency of the biotin labeling reaction was verified using NeutrAvidin (10mg/mL) with a gel-shift assay.
|
| |
|
| Hybridization protocol |
Samples were injected into arrays and placed in the Affymetrix Genechip® Hybridization Oven 640 at 45° C and 60 RPM for 16-18 hours overnight.
|
| Scan protocol |
Arrays were stained using the Affymetrix Genechip® Fluidics Station 450 and scanned with the Affymetrix Genechip® Scanner 3000.
|
| Description |
Total RNA from Male WT (p38a-f/f) macrophages; pooled from 2-3 samples from individual mice, biological replicate 3
|
| Data processing |
The signal intensity for each probe on each chip was calculated from scanned images using GeneChip Operating Software (Affymetrix), and signal intensities were analyzed using BioConductor (http://www.bioconductor.org). Probe intensities were background corrected, normalized, and summarized using the Robust Multichip Average method described by Speed and coworkers (Bolstad et al., 2003; Irizarry et al., 2003). An alternative normalization method based on housekeeping genes did not significantly change the results. Probe sets were considered differentially expressed when the signed fold change was ≥ 1.5, and the P-value was < 0.05.
MoGene-2_0-st.pgf
MoGene-2_0-st.mps
|
| |
|
| Submission date |
Oct 25, 2013 |
| Last update date |
Nov 01, 2013 |
| Contact name |
Dimitry Krementsov |
| E-mail(s) |
dkrement@uvm.edu
|
| Organization name |
University of Vermont
|
| Department |
BHSC
|
| Street address |
312C Rowell bldg, 106 Carrigan drive
|
| City |
89 Beaumont ave |
| State/province |
VT |
| ZIP/Postal code |
05405 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE51707 |
Sex-specific control of CNS autoimmunity by p38 MAPK signaling in myeloid cells |
|
