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Sample GSM2829255 Query DataSets for GSM2829255
Status Public on Jun 04, 2020
Title DT3
Sample type RNA
 
Source name Resolving Lung Type II alveolar epithelial cells in absence of Tregs
Organism Mus musculus
Characteristics tissue: Type II alveolar epithelial cells
genotype: Foxp3DTR C57BL/6 background
age: 8-12 weeks
Treatment protocol Anesthetized mouse strains C57BL/6, Foxp3EGFP (C57BL/6 background), or Foxp3DTR (C57BL/6 background) received Escherichia coli LPS O55:B5 (3mg/kg) instilled intratracheally into the lungs. Type II alveolar epithelial cells from single cell suspension from the lungs were enriched by depleting CD31+ and CD45+ cells before sorted using a FACSAria (Becton Dickinson, San Jose, CA). Briefly, we utilized two transgenic strains, (Foxp3EGFP; Foxp3DTR Jackson Laboratory) to study differential transcript expression of type II alveolar epithelial cells in the presence (Foxp3EGFP) or absence (Foxp3DTR) of Foxp3+ Tregs. Foxp3EGFP mice express green fluorescent protein (GFP) downstream of the endogenous Foxp3 stop codon, resulting in fluorescence of all Foxp3+ Tregs. The Foxp3DTR mice express the human diphtheria toxin receptor (DTR) along with GFP, whose genes have been inserted into the 3’ untranslated region of the Foxp3 locus. These mice allow specific elimination of Foxp3+ Tregs in vivo through intraperitoneal (i.p.) administration of diphtheria toxin (DT). This results in apoptosis of Treg without significant systemic toxicity. By examining the effects of Treg depletion in the presence of other lymphocytes, we can better understand the cellular interactions that govern resolution of lung injury. At 7 days post LPS-induced injury, single cell suspensions were prepared from enzymatically digested and dissected mouse lungs from these three strains. The cells were washed with PBS containing 2 mM EDTA and 1.5% bovine serum albumin (FACS buffer). The cells were filtered through a 100 mM filter, and then the filter samples were subjected to negative selection by depleting CD31+ and CD45+ cells using MojoSort biotin nanobeads per manufacturer's protocols (Biolegend, San Diego, CA). The CD31-CD45- cell populations were resuspended in FACS buffer at a concentration of 2 x 107 cells/mL, and then subjected to cell sorting for CD326+ MHCII+CD104-CD24-T1a- cells, which are type II alveolar epithelial 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 mL 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 mL 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 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 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 Resolving AT2 depleted Tregs rep3
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.
 
Submission date Oct 24, 2017
Last update date Jun 04, 2020
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)
GSE106081 Transcriptional Analysis of Type II Alveolar Epithelial Cells in the Presence or Absence of Foxp3+ Regulatory T Cells during Experimental Acute Lung Injury Resolution

Data table header descriptions
ID_REF
VALUE RMA log2

Data table
ID_REF VALUE
AB618486 1.9519
AF067064 1.88839
AF075717 2.18309
AF143094 4.22284
AF248058 8.75478
AF285583 2.35059
AF295105 3.29717
AF358728 2.32857
AF394949 2.62668
AF469169 2.19046
AJ237586 2.05705
AJ459773 3.1904
AK002956 8.19474
AK003851 2.234
AK005807 1.94207
AK005827 2.01618
AK006077 1.96293
AK006189 1.55872
AK006298 3.0948
AK006338 2.4547

Total number of rows: 31351

Table truncated, full table size 759 Kbytes.




Supplementary file Size Download File type/resource
GSM2829255_DT3.CEL.gz 4.2 Mb (ftp)(http) CEL
Processed data included within Sample table
Processed data are available on Series record

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