|
| Status |
Public on Feb 12, 2019 |
| Title |
Control Lung Treg rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Control Lung Tregs
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: Foxp3EGFP
age: 8-12 weeks
cell type: Lung Regulatory T Cells
treatment: uninjured
|
| Treatment protocol |
Anesthetized mice, Foxp3EGFP (C57BL/6 background), received Escherichia coli LPS O55:B5 (3mg/kg) instilled intratracheally into the lungs. Lymphocytes from single cell suspension from either the lung or spleen were enriched by Percoll density centrifugation (Sigma-Aldrich, St. Louis, MO) and CD4+ GFP+ Tregs then were sorted using a FACSAria (Becton Dickinson, San Jose, CA). Briefly, single cell suspensions were prepared from enzymatically digested and dissected mouse lungs or mechanical dissociation of mouse spleens. The cells were washed with PBS containing 2 mM EDTA and 1.5% bovine serum albumin (FACS buffer). The cells were filtered through a 40 mM filter, and then the filter samples were centrifuged in a 33% Percoll gradient at 2,000 rpm with slow acceleration and slow deceleration to pellet the lymphocyte population. The lymphocyte population were resuspended in FACS buffer at a concentration of 2 x 10e7 cells/mL before then cell sorting CD4+ GFP+ cells. Sorted cells were collected for mRNA analysis. Cells were kept at 4-8°C during staining and sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Zymo Direct-zol RNA MiniPrep with TRI Reagent miRNeasy kits from Zymo Research (Irvine, CA, USA) according to the manufacturer's instruction with the following modifications: 1) snap-frozen cells were lysed in at least 125 uL of TRI reagent without thawing cells; 2) cells were not centrifuged to collect debris or DNaseI-treated before applying to column. All samples were eluted in 25 uL of DNase/RNase-Free water.
|
| Label |
biotin
|
| Label protocol |
Total RNA (225 ng) was used to synthesize fragmented and labeled sense-strand cDNA and hybridize onto Affymetrix 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 Affymetrix peg plate arrays was carried out using the Affymetrix GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
|
| Scan protocol |
Scanning of the Affymetrix peg plate arrays was carried out using the Affymetrix GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
|
| Description |
Treg Control3
mRNA expression data from mouse lung regulatory T cells.
|
| 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 < 0.1, and fold change > 1.5, 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 to identify significant Gene Ontology (GO) biological processes.
Custom gene annotation, or alternative mapping of Affymetrix probesets, based on Affymetrix annotation version NA35 and ENSEMBL gene annotation v81 was used.
|
| |
|
| Submission date |
Sep 20, 2017 |
| Last update date |
Feb 12, 2019 |
| Contact name |
Jason Robert Mock |
| E-mail(s) |
jason_mock@med.unc.edu
|
| Phone |
9199625347
|
| Organization name |
University of North Carolina
|
| Department |
Internal Medicine
|
| Street address |
125 Mason Farm Rd, Marsico Hall 7229G
|
| City |
Chapel Hill |
| State/province |
North Carolina |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platform ID |
GPL22070 |
| Series (1) |
| GSE104088 |
Transcriptional Analysis of Foxp3+ Regulatory T Cells in Experimental Acute Lung Injury Resolution |
|
