|
| Status |
Public on Sep 09, 2016 |
| Title |
WT neutrophils SP rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Lung neutrophils isolated from WT mice given S. pneumoniae
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
background strain: C57BL/6
cell type: neutrophils
genotype: Wild type (WT)
age: 6-12 weeks
inoculation agent: Streptococcus pneumoniae
|
| Treatment protocol |
Anesthetized mice received PBS or a suspension containing S. pneumoniae instilled intratracheally into the left lung at a dose of 2.3 µl/g mouse body weight. Neutrophils were isolated using MACS beads (Miltenyi, Auburn, CA) according to the manufacturer’s instructions. Briefly, single cell suspensions were prepared from dissected mouse lungs. The cells were washed with PBS containing 2 mM EDTA and 0.5% bovine serum albumin, and incubated with biotin-anti-Ly6G and magnetic beads coated with anti-biotin. The samples were passed through a column in a magnetic field, and cells bound to beads were collected for further analysis. Cells were kept at 4-8°C during staining and magnetic bead isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using miRNeasy kits from Qiagen (Valencia, CA, USA) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Total RNA (250 ng) was used to synthesize fragmented and labeled sense-strand cDNA and hybridize onto Affymetrx arrays. The Affymetrix HT WT User Manual was followed to prepare the samples. Briefly, the WT Expression HT Kit for Robotics (Ambion) was used to generate sense-strand cDNA from total RNA. Following synthesis of sense-strand cDNA, the cDNA was fragmented and labeled with the Affymetrix GeneChip HT Terminal Labeling Kit. The Beckman Coulter Biomek FXP Laboratory Automation Workstation with the Target Express set up was used to prepare the samples with these two kits.
|
| |
|
| Hybridization protocol |
Fragmented and labeled cDNA was used to prepare a hybridization cocktail with the Affymetrix GeneTitan Hybridization Wash and Stain Kit for WT Arrays. Hybridization, washing, staining and scanning of the Affymtrix peg plate arrays was carried out using the Affymetrin GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
|
| Scan protocol |
Scanning of the Affymtrix peg plate arrays was carried out using the Affymetrin GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
|
| Description |
Gene expression data from mouse lung neutrophils
|
| Data processing |
Affymetrix Expression Console Software was used for basic data analysis and quality control. Microarray data were preprocessed by RMA (Robust Multiarray Average) background correction, GC content and sequence correction, quantile normalization, and median polish summarization, and expression levels compared using ANOVA on log2 intensities to identify transcripts that were differentially expressed between genotype and treatment groups. Differentially expressed (DE) mRNAs were filtered at Benjamini-Hochberg FDR < 0.05, and fold change > 2, and expression patterns of DE transcripts were visualized using hierarchical clustering. Expression analyses were performed using Partek Genomics Suite (Partek Inc., St. Louis. MO, USA). Gene Set Enrichment Analysis (GSEA) was performed according to the method described (12, 13). In addition to gene sets derived from Gene Ontology (GO) biological processes, GSEA for Nrf2 regulated genes was performed using lists of Nrf2 regulated genes from the literature (14, 15).
The .mps file used in this analysis is available on the Superseries record.
|
| |
|
| Submission date |
Jun 22, 2016 |
| Last update date |
Sep 09, 2016 |
| Contact name |
Claire M. Doerschuk |
| Organization name |
University of North Carolina at Chapel Hill
|
| Department |
Marsico Lung Institute
|
| Street address |
Marsico Hall 7205 CB#7248
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7248 |
| Country |
USA |
| |
|
| Platform ID |
GPL22070 |
| Series (2) |
| GSE83613 |
Gene expression profiling of neutrophils and whole lung tissue from wildtype and Nrf2 null (Nfe2l2-/-) mice during S. pneumoniae pneumonia [neutrophils] |
| GSE83615 |
Gene expression profiling of neutrophils and whole lung tissue from wildtype and Nrf2 null (Nfe2l2-/-) mice during S. pneumoniae pneumonia |
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