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Sample GSM2789652 Query DataSets for GSM2789652
Status Public on Feb 12, 2019
Title Resolving Splenic Treg rep1
Sample type RNA
 
Source name Resolving Splenic Tregs
Organism Mus musculus
Characteristics strain/background: C57BL/6
genotype/variation: Foxp3EGFP
age: 8-12 weeks
cell type: Splenic Regulatory T Cells
treatment: lipopolysaccharide-induced lung injury
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 Spleen1
mRNA expression data from mouse splenic 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

Data table header descriptions
ID_REF
VALUE RMA log2

Data table
ID_REF VALUE
AB618486 1.96473
AF067064 2.00686
AF075717 2.42538
AF143094 6.06977
AF248058 2.6072
AF285583 2.27475
AF295105 2.2453
AF358728 2.12841
AF394949 2.78893
AF469169 2.27957
AJ237586 1.99857
AJ459773 4.97644
AK002956 7.74137
AK003851 2.1562
AK005807 2.25564
AK005827 2.23653
AK006077 1.71991
AK006189 4.78012
AK006298 2.12071
AK006338 2.45362

Total number of rows: 31351

Table truncated, full table size 637 Kbytes.




Supplementary file Size Download File type/resource
GSM2789652_Treg_Spleen1.CEL.gz 4.4 Mb (ftp)(http) CEL
Processed data included within Sample table

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